![3.1.4. Assessment of
RNA Quality
Initially, it may be necessary to check batches of RNA for signs of
RNAse contamination, which leads to degradation of the RNA.
Commonly this can be achieved by running an aliquot of RNA on a
denaturing agarose gel, checking for the presence of a single species
of defined size without a smear of lower-molecular-weight frag-
ments, indicative of RNA degradation. However, due to the het-
erogeneity in size of the poly-A tail, a single species is rarely seen,
leading to some degree of uncertainty about the quality of the
RNA. Instead, we check that the RNA can be translated into a
protein of the predicted molecular weight. This is achieved using
1–2 mg of RNA in a Rabbit Reticulocyte Lysate reaction (Promega).
We label the protein with [35
S]-Pro-Mix, and, following separation
on SDS-PAGE, use autoradiography to determine its molecular
weight. Alternatively, we have also satisfactorily used ‘‘cold’’
methionine in the reaction, and then used antibodies to detect
the protein of interest on a western blot following the SDS-PAGE.
3.2. Microinjection of
Eggs
1. Zona intact mouse eggs are placed in a shallow drop (1 ml) of
media covered in oil, in the lid of a petri dish. The dish is placed
on the microscope stage without heating. A silver wire is placed
in the injection drop and this wire is held in place via a small
manipulator. This wire is connected to a longer standard cop-
per wire to the chassis ground of the electrical amplifier. This
wire allows for an electrical circuit to form between the ground
and the tip of the pipette once it is place in the media (a circuit
is indicated by the amplifier). If this does not occur, then the
pipette should be replaced because the tip is probably blocked.
2. The RNA solution to be injected should be spun (13,000 rpm
in a benchtop microcentrifuge) for several minutes before
injection. For RNA injection, we generally use tips of
0.75–0.9 mm in diameter. The injection pipettes are backfilled
with 1 ml of injection solution containing the RNA.
3. This pipette is then held in the specialized holder (containing a
silver wire) that is then fitted onto the preamplifier that is itself
clamped into the micromanipulator.
4. For experiments where fluorescence is also measured, to look
at changes in Ca2+
dynamics within the egg, the luciferase-
tagged cRNA is mixed with an equal volume of 1-mM OGBD
prepared in KCl Hepes pH 7.2. Even if a Ca2+
dye is not to be
used, it is useful to mix the RNA solution 1:1 with KCL Hepes
buffer so as to have some salts present in the injection buffer.
5. For injection, each egg is held by suction with the holding
pipette and then the tip of the injection pipette is manipulated
so that it will touch the plasma membrane. The injection
pipette is then pushed into egg in a way that deforms the
zona pellucida. At some point, the zona will jump back into
Luciferase Tag Expression in Mouse Eggs 23](https://image.slidesharecdn.com/1243661-250518040736-6c00b86d/85/Microinjection-Methods-And-Applications-1st-Edition-Jill-C-Sible-41-320.jpg)
![3. Kouchi, Z., Fukami, K., Shikano, T., Oda,
S., Nakamura, Y., Takenawa, T and Miya-
zaki, S. (2004) Recombinant phospholipase
Czeta has high Ca2þ
sensitivity and induces
Ca2þ
oscillations in mouse eggs. J. Biol.
Chem. 279, 10408–12.
4. Nomikos, M., Blayney, L.M., Larman, M.G.,
Campbell, K., Rossbach, A., Saunders, C.M.,
Swann, K. and Lai, F.A. (2005) Role of phos-
pholipase C-(zeta) domains in Ca2+
-depen-
dent phosphatidylinositol 4,5-bisphosphate
hydrolysis and cytoplasmic Ca2+
oscillations.
J. Biol. Chem. 280, 31011–18.
5. Dumollard, R., Marangos, P., Fitzharris, G.,
Swann, K., Duchen, M. and Carroll, J.
(2004) Sperm-triggered [Ca2+
] oscillations
and Ca2+
homeostasis in the mouse egg
have an absolute requirement for mitochon-
drial ATP production. Development 131,
3057–67.
6. Yoda, A, Oda, S., Shikano, T., Kouchi, Z.,
Awaji, T., Shirakawa, H., Kinoshita, K and
Miyazaki, S. (2004) Ca2+
oscillation-indu-
cing phospholipase C zeta expressed in
mouse eggs is accumulated in the pronucleus
during egg activation. Devel. Biol. 268,
245–57
7. Cox, L.J., Larman, M.G., Saunders, C.M.,
Hashimoto, K., Swann, K. and Lai, F.A.
(2002) Sperm phospholipase C from
humans and cynomolgus monkeys triggers
Ca2+
oscillations, activation and develop-
ment of mouse oocytes. Reproduction 124,
611–23.
8. Summers, M.C., Bhatnagar. P.R., Lawitts.
J.A., Biggers. J.D. (1995) Fertilization in
vitro of mouse ova from inbred and outbred
strains: complete preimplantation embryo
development in glucose-supplemented
KSOM. Biol. Reprod. 53, 431–7.
9. Summers, M.C., McGinnis, L.K., Lawitts,
J.A., Raffin, M., Biggers, J.D. (2000) IVF
of mouse ova in a simplex optimized medium
supplemented with amino acids. Hum.
Reprod. 15, 1791–801.
10. Hogan, B., Costantini, F. and Lacy, E.
(1986). Manipulating the Mouse Embryo: A
Laboratory Manual. Cold Spring Harbour
Laboratory, Cold Spring Harbor, NY.
11. Schnorf, M., Potrykus, I. and Neuhaus, G.
(1994) Microinjection technique: routine
system for characterization of microcapil-
laries by bubble pressure measurement. Exp.
Cell. Res. 210, 260–7.
12. Campbell, A.K. (1988) ‘Chemiluminescence’
Ellis Horwood Series on Biomedicine.
13. Sambrook, J. and Russell, D.W. (2001)
Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY.
14. Campbell, K. and Swann, K. (2006) Ca2+
oscillations stimulate an ATP increase during
fertilization. Devel. Biol. 298, 225–53.
15. Carroll, J., Swann, K., Whittingham, D. and
Whitaker, M.J. (1994) Spatiotemporal
dynamics of intracellular [Ca2+
]i oscillations
during growth and meiotic maturation of
mouse oocytes. Development 120, 3507–17.
Luciferase Tag Expression in Mouse Eggs 29](https://image.slidesharecdn.com/1243661-250518040736-6c00b86d/85/Microinjection-Methods-And-Applications-1st-Edition-Jill-C-Sible-47-320.jpg)
![thousands, I’d still be thief;” such were the words uttered by a youth
in Coldbath-fields Prison, and overheard by the governor.[49]
If the machinery for preventing and detecting crime has so vastly
improved within this present century, the same may be said for the
method of dispensing justice. Up to as late as 1792, the magistrates
of Bow-street—the first “police-office,” as it was then termed—were
paid in that most obnoxious of all modes, by fees, which were often
obtained in a manner so disgraceful that the magistrates got the
name of “trading justices” and “basket justices.” Our old friend John
Townsend, whom we must summon once more to our aid, gives an
insight into their proceedings, and he knew them well. He said, “The
plan used to be to issue warrants, and to take up all the poor devils
in the streets, and then there was the bailing them, 2s. 4d., which
the magistrate had. In taking up a hundred girls, that would make,
at 2s. 4d., 11l. 13s. 4d. They sent none to jail, for the bailing them
was so much better!” The old Bow-street worthy then draws a
picture of the magistrate settling the amount of these ill-gotten fees
with his clerk on the Monday morning. The “basket justices” were so
called, because they allowed themselves to be bought over by
presents of baskets of game. These enormities were so glaring, that,
according to Townsend, “they at last led to the Police Bill, and it was
a great blessing to the public to do away with these men, for they
were nothing better than the encouragers of blacklegs, vice, and
plunderers. There is no doubt about it.” In 1792 seven other
“offices” were established, namely, Queen-square, Great
Marlborough-street, Hatton Garden, Worship-street, Lambeth,
Shadwell, and Union-street, each office having three magistrates,
who did the duties alternately. These, by the addition of the
suburban courts, have since been augmented to eleven. They form
the judgment-seats to which all offenders in this great capital of
2,500,000 inhabitants are brought, either to be punished summarily,
or to be remanded to the sessions to take their trial.
The police-courts may be likened to so many shafts sunk in the
smooth surface of society, through which the seething mass of](https://image.slidesharecdn.com/1243661-250518040736-6c00b86d/85/Microinjection-Methods-And-Applications-1st-Edition-Jill-C-Sible-74-320.jpg)
Microinjection Methods And Applications 1st Edition Jill C Sible
- 1. Microinjection Methods And Applications 1st Edition Jill C Sible download https://ebookbell.com/product/microinjection-methods-and- applications-1st-edition-jill-c-sible-2487322 Explore and download more ebooks at ebookbell.com
- 2. Here are some recommended products that we believe you will be interested in. You can click the link to download. Microinjection Methods And Protocols Hardcover Chengyu Liu Yubin Du https://ebookbell.com/product/microinjection-methods-and-protocols- hardcover-chengyu-liu-yubin-du-7260364
- 5. M E T H O D S I N M O L E C U L A R B I O L O G Y TM John M. Walker, SERIES EDITOR 518. Microinjection: Methods and Applications, edited by David J. Carroll, 2009 502. Bacteriophages: Methods and Protocols, Volume 2: Mole- cular and Applied Aspects, edited by Martha R. J. Clokie and Andrew M. Kropinski, 2009 501. Bacteriophages: Methods and Protocols, Volume 1: Isola- tion, Characterization, and Interactions, edited by Martha R. J. Clokie and Andrew M. Kropinski, 2009 496. DNA and RNA Profiling in Human Blood: Methods and Protocols, edited by Peter Bugert, 2009 493. Auditory and Vestibular Research: Methods and Proto- cols, edited by Bernd Sokolowski, 2009 490. Protein Structure, Stability, and Interactions, edited by John W. Shriver, 2009 489. Dynamic Brain Imaging: Methods and Protocols, edited by Fahmeed Hyder, 2009 485. HIV Protocols: Methods and Protocols, edited by Vinayaka R. Prasad and Ganjam V. Kalpana, 2009 484. Functional Proteomics: Methods and Protocols, edited by Julie D. Thompson, Christine Schaeffer-Reiss, and Marius Ueffing, 2008 483. Recombinant Proteins From Plants: Methods and Protocols, edited by Lóic Faye and Veronique Gomord, 2008 482. Stem Cells in Regenerative Medicine: Methods and Protocols, edited by Julie Audet and William L. Stanford, 2008 481. Hepatocyte Transplantation: Methods and Protocols, edited by Anil Dhawan and Robin D. Hughes, 2008 480. Macromolecular Drug Delivery: Methods and Proto- cols, edited by Mattias Belting, 2008 479. Plant Signal Transduction: Methods and Protocols, edi- ted by Thomas Pfannschmidt, 2008 478. Transgenic Wheat, Barley and Oats: Production and Characterization Protocols, edited by Huw D. Jones and Peter R. Shewry, 2008 477. Advanced Protocols in Oxidative Stress I, edited by Donald Armstrong, 2008 476. Redox-Mediated Signal Transduction: Methods and Protocols, edited by John T. Hancock, 2008 475. Cell Fusion: Overviews and Methods, edited by Eliza- beth H. Chen, 2008 474. Nanostructure Design: Methods and Protocols, edited by Ehud Gazit and Ruth Nussinov, 2008 473. Clinical Epidemiology: Practice and Methods, edited by Patrick Parfrey and Brendon Barrett, 2008 472. Cancer Epidemiology, Volume 2: Modifiable Factors, edited by Mukesh Verma, 2008 471. Cancer Epidemiology, Volume 1: Host Susceptibility Factors, edited by Mukesh Verma, 2008 470. Host-Pathogen Interactions: Methods and Protocols, edited by Steffen Rupp and Kai Sohn, 2008 469. Wnt Signaling, Volume 2: Pathway Models, edited by Elizabeth Vincan, 2008 468. Wnt Signaling, Volume 1: Pathway Methods and Mammalian Models, edited by Elizabeth Vincan, 2008 467. Angiogenesis Protocols: Second Edition, edited by Stewart Martin and Cliff Murray, 2008 466. Kidney Research: Experimental Protocols, edited by Tim D. Hewitson and Gavin J. Becker, 2008 465. Mycobacteria, Second Edition, edited by Tanya Par- ish and Amanda Claire Brown, 2008 464. The Nucleus, Volume 2: Physical Properties and Ima- ging Methods, edited by Ronald Hancock, 2008 463. The Nucleus, Volume 1: Nuclei and Subnuclear Com- ponents, edited by Ronald Hancock, 2008 462. Lipid Signaling Protocols, edited by Banafshe Lari- jani, Rudiger Woscholski, and Colin A. Rosser, 2008 461. Molecular Embryology: Methods and Protocols, Second Edition, edited by Paul Sharpe and Ivor Mason, 2008 460. Essential Concepts in Toxicogenomics, edited by Donna L. Mendrick and William B. Mattes, 2008 459. Prion Protein Protocols, edited by Andrew F. Hill, 2008 458. Artificial Neural Networks: Methods and Applications, edited by David S. Livingstone, 2008 457. Membrane Trafficking, edited by Ales Vancura, 2008 456. Adipose Tissue Protocols, Second Edition, edited by Kaiping Yang, 2008 455. Osteoporosis, edited by Jennifer J.Westendorf, 2008 454. SARS- and Other Coronaviruses: Laboratory Protocols, edited by Dave Cavanagh, 2008 453. Bioinformatics, Volume 2: Structure, Function, and Applications, edited by Jonathan M. Keith, 2008 452. Bioinformatics, Volume 1: Data, Sequence Analysis, and Evolution, edited by Jonathan M. Keith, 2008 451. Plant Virology Protocols: From Viral Sequence to Pro- tein Function, edited by Gary Foster, Elisabeth Johan- sen, Yiguo Hong, and Peter Nagy, 2008 450. Germline Stem Cells, edited by Steven X. Hou and Shree Ram Singh, 2008 449. Mesenchymal Stem Cells: Methods and Protocols, edi- ted by Darwin J. Prockop, Douglas G. Phinney, and Bruce A. Brunnell, 2008 448. Pharmacogenomics in Drug Discovery and Develop- ment, edited by Qing Yan, 2008 447. Alcohol: Methods and Protocols, edited by Laura E. Nagy, 2008 446. Post-translational Modifications of Proteins: Tools for Functional Proteomics, Second Edition, edited by Christoph Kannicht, 2008 445. Autophagosome and Phagosome, edited by Vojo Deretic, 2008 444. Prenatal Diagnosis, edited by Sinhue Hahn and Laird G. Jackson, 2008 443. Molecular Modeling of Proteins, edited by Andreas Kukol, 2008
- 6. M E T H O D S I N M O L E C U L A R B I O L O G Y TM Microinjection Methods and Applications Edited by David J. Carroll Florida Institute of Technology, Melbourne, FL, USA
- 7. Editor David J. Carroll Florida Institute of Technology Melbourne, FL USA dcarroll@fit.edu Series Editor John M. Walker University of Hertfordshire Hatfield, Hert. UK ISSN: 1064-3745 e-ISSN: 1940-6029 ISBN: 978-1-58829-884-3 e-ISBN: 978-1-59745-202-1 DOI 10.1007/978-1-59745-202-1 Library of Congress Control Number: 2008938642 # Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer ScienceþBusiness Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Cover illustration: The image shows how a holding pipet is used to immobilize a zebrafish oocyte for microinjection. A typical holding pipet produced by flame polishing of a glass capillary is used to pick up and immobilize an unfertilized oocyte. Magnification is indicated by the bar which represents 100mm. Photo credit: William H. Kinsey. Printed on acid-free paper springer.com
- 8. Preface David J. Carroll Abstract Cellular microinjection techniques have developed over the last century along with the evolution of other biological fields. For example, developmental biologists have used a variety of microinjection techniques to transfer cytoplasm between cells, inject antibodies and peptides, and express foreign genes in specific tissues to advance their understanding of cell specification and determination. Molecular biologists finely craft glass microneedles and tools for the manipulation of cells in order to study gene expression and communication between cells. As these techniques have matured, microinjection has become accessible and, thus, exploited by a larger segment of the scientific and medical community. As more and more information is gathered from the various genome projects, demand grows for methods to validate these new data. Microinjection can help address this need. In particular, microinjection has proven valuable for the confirmation and extension of in vitro results in an in vivo setting – the living cell. The technique has also found a home in the clinical setting, most obviously within the community of fertility specialists for in vitro fertilization methods and for those excited about the possibility of therapeutic cloning. This book explores the use of microinjection for a wide range of scientific uses. There are special considerations for each application of microinjection – whether it is the injection of antibodies, fusion proteins, DNA, and in vitro-synthesized RNA or the production of transgenic animals. It is hoped that these methods will be of interest for all biologists for use in the research laboratory, as well as for clinicians interested in applying this powerful method for treatment in the clinic. 1. A Brief History of Microinjection The technique of microinjection was born of necessity and owes its history to a combination of fields. Credit for the initial descrip- tion of a coherent microinjection technique could be given to Marshall Barber, who developed methods for producing fine glass capillary pipettes for isolating and manipulating single bacterial cells (1,2), or to the embryologist Laurent Chabry, credited with developing the glass microcapillary and micromanipulator as tools for his studies on teratology in ascidian blastomeres (3,4). Barber incorporated techniques into his injection method that are still used today, including the first use of mercury for controlling the movement of small volumes of fluid (see below) and the use of a second pipette to hold the cells as the injection is completed (5,6). The technique of microinjection moved from being a useful method practiced by a few resourceful scientists to an exciting main- stream application when Gurdon and colleagues demonstrated that v
- 9. purified mRNA from one cell could be injected into the cytoplasm of another cell and actually get translated into protein (7). This was remarkable for several reasons: (1) the mRNA was stable, at least stable enough that it was capable of directing the synthesis of detectable levels of protein; (2) no special factors, other than the exogenous mRNA itself, were required for this to work; and (3) success was independent of the cell type of the donor RNA. In fact, species differences do not appear to matter, as they were able to obtain the synthesis of rabbit hemoglobin in Xenopus laevis oocytes. Since then, the method of expressing protein from microin- jected mRNA has been used to great advantage in studies for developmental biology, neurobiology, cell biology, and signal transduction, just to name a few. An excellent overview of this method is given by Douglas Melton (8), in which he explains why the oocyte (and the Xenopus oocyte, in particular) provides the perfect system for this to work, along with describing details of the current methods at that time. One great advantage of this system, compared to translation in vitro or by induced expression in bacteria, is that the mRNA is placed in a living eukaryotic cell so the protein is processed properly and subject to post-translational modification. Of course, the technique is not only useful for microinjecting RNA into Xenopus oocytes. It has been utilized for transferring cytoplasm from one cell to another, such as was done in the experiments that led to the discovery that maturation promoting factor (MPF) was neither species nor cell-type specific (9–11). This fundamental work provided the groundwork for the eventual discovery of cyclins and cyclin-dependent kinases (12,13). Pro- teins have been injected into cells for the study of cell structure and function. One of the first examples of this utility was the microinjection of fluorescently labeled -actinin into living fibro- blasts, allowing the visualization of this molecule integrating dynamically into the cytoskeleton (14). Of course, the concept of following labeled proteins in a living cell has since exploded with the availability of green fluorescent protein (GFP) vectors and protocols (15,16). One tremendous advantage of microinjection, when com- pared to other methods of introducing material into cells, such as electroporation or chemical membrane permeabilization, is the ability to be quantitative. Methods for quantitative microinjection have developed over the years, but began in earnest with a study by Hiramoto on the process of fertilization (17). In that study, live spermatozoa were microinjected into sea urchin eggs to develop an assay for the identification of a ‘‘substance or substances which trigger the train of fertilization reactions in the egg’’. No such substance was identified in that study (see Chapter 2), but meth- ods to precisely control and quantify the volume of the injection using a mercury-filled needle connected to a screw-controlled vi Preface
- 10. syringe were described. Hiramoto’s basic method has been further refined (18–20), but is still being used today. This book provides methods of microinjection coupled with modern molecular techniques, such as RNAi, morpholino anti- sense oligonucleotides, GFP expression, or the production of transgenic cells or animals, for example. However, the book also revisits classic uses of microinjection, such as mRNA expression or nuclear transfer, with modern twists. 2. Book Content The classic technique of microinjecting Xenopus zygotes with mRNA prepared in vitro is revisited in Chapter 1 with a focus on studying proteins involved in the cell cycle. A discussion of the use of microinjection compared to other methods of manipulating proteins in a living cell is given. The chapter also provides con- sideration of choosing the appropriate plasmid vector with an eye toward downstream analysis of the effectiveness of the protein expression. This discussion should be very useful for investigators considering the use of mRNA microinjection for the first time. In Chapter 2, a very clever method using a luciferase chimera to visualize the expression of a protein, PLC, that causes calcium release and egg activation during fertilization in mammals is described (21). Methods are given for producing the luciferase- labeled cRNA, along with techniques for assessing the expression of the fusion protein by microscopy, which allows simultaneous imaging of fluorescent indicators (e.g., a Ca2+ indicator), or by luminometer for quantification of luciferase expression. This method could easily be applied to other molecules in any cell type that will express exogenous RNA. In particular, it promises to be useful for combining detection of low levels of protein expression with quantification of those molecules from a reason- able number of cells. In Chapter 3, a very straightforward technique for using antisense morpholino oligos to specifically remove 14-3-3 pro- teins in Xenopus laevis is described. Morpholino oligos are non- ionic DNA analogs that possess an altered phosphodiester backbone. This apparently makes them more resistant to nucleases and, because they are not charged, less likely to interact nonspe- cifically with proteins (22). This technique complements their earlier work in which the 14-3-3 proteins were studied using peptide inhibitors or dominant-interfering GST fusion proteins (23). Their method utilizes morpholinos that target the initiation codon and the 22 ribonucleotides immediately downstream, Preface vii
- 11. which may produce more effective inhibition. This chapter out- lines methods for (1) procuring the Xenopus sperm and eggs, (2) setting up and using a pressure microinjection apparatus with the Xenopus one- or two-cell embryo, (3) for analyzing and quantify- ing protein levels by western blotting, and (4) for analyzing the phenotypic effects of the morpholino injections. An alternate strategy for morpholino design that targets the 50 untranslated region of the target gene is also discussed. Methods for microinjecting peptides and fusion proteins into Xenopus laevis oocytes are given in Chapter 4. Following a very clear description of ovary dissection and oocyte procurement by collagenase treatment, the procedure for performing rapid micro- injections into the Xenopus oocyte is given. Accompanied by excellent photographs, this chapter explains the process in detail from start to finish. For example, in the text and in the Notes section, suggestions are given regarding the exact size for the tip of the microinjection needle and when the needle should be changed, the maximum volume and concentration of protein that should be injected into an individual oocyte, and how to deal with multiple injections into the same oocyte. This type of detail fills the chapter and will help other investigators maximize success when they adopt this method. A method to combine microinjection with western blot ana- lysis is described in Chapter 5. In this procedure, single oocytes are microinjected with a pharmacological inhibitor of RAS and then assayed for the presence of phosphorylated mitogen-acti- vated protein kinase (pMAPK), which indicates an active enzyme, by immunoblotting. Thus far, this has been applied only for the analysis of the pMAPK during oocyte maturation and fertilization in starfish oocytes. However, it should also be useful in other large cells (such as Xenopus oocytes) and, as detection methods improve, it could easily be adapted to the analysis of other proteins in other systems. Analysis of single cells should prove useful because it eliminates the variability inherent to the analysis of cell populations. This book considers the use of many different model systems, including the zebrafish Danio rerio. The zebrafish has proven extremely useful for experimental study because it is amenable to genetic studies, the embryo develops rapidly, and it is optically clear (you can see inside). The zebrafish zygote and early embryo have been utilized as a system for microinjection because of the large size of the eggs and the ease with which the adults are main- tained. However, for a variety of reasons it would be advantageous to inject the mature egg prior to fertilization and this has proven difficult. In Chapter 6, a practical method for microinjection, and subsequent insemination, of the unfertilized zebrafish egg is reported. This will open up more opportunities for exploiting an already useful model system. viii Preface
- 12. In the first method (Chapter 7) to deal with a ‘non-gamete’ system, techniques for antibody microinjection and oligofecta- mine transfection of RNAi for analysis of protein function in living tissue culture cells are compared. The chapter is very detailed which should allow these methods to be adapted to many different situations. The chapter also features a very extensive and useful notes section, detailing specifics of each of the methods. Methods for developing recombinant cells lines by microin- jection into the nucleus are presented in Chapter 8. This very interesting article demonstrates that introduction of plasmid DNA for the GFP, in several cell types, leads to stable transduction as assayed by flow cytometry of GFP fluorescence up to 1 month after microinjection! The chapter details methods that could be adapted to virtually any cell type and for any DNA. Conditions for optimizing the production of the transformed cells lines are tested in this chapter and detailed notes are given to help any investigator interested in attempting this method. Different experimental methods, including transposons, I-SceI meganuclease, and direct injection of linearized DNA, have been used to produce transgenic Xenopus for the study of a variety of problems (24–26). However, these methods rely upon random insertion into the Xenopus genome or produce multiple copies. In Chapter 9, a technique is described for the targeted insertion of a single copy of the gene of interest into Xenopus laevis using phiC31 integrase. By incorporating insulator sequences into the plasmid design, they improve expression from the reporter gene making this method extremely useful for anyone wishing to express a transgene at approximately endogenous levels. Transgenics are also the topic of Chapter 10. A method for producing transgenic Caenorhabditis elegans by microinjecting DNA directly into the hermaphrodite gonad is provided. For those not familiar with C. elegans, this chapter provides an excel- lent introduction to this powerful system. It recounts the basic reproductive biology of the worm and includes a discussion of applications for microinjection. Techniques for cultivating the worms and special hints for maximizing success of microinjection are explained in detail. One of the great advantages of using microinjection to intro- duce molecules into living cells is the ability to perform quantita- tive experiments. Chapter 11 gives detailed methods for the quantitative microinjection of picoliter quantities into mouse oocytes and eggs in dishes on an inverted microscope. The tech- nique is described in wonderful detail and covers all aspects from making the glass bottom dishes that hold the oocytes to the injection process itself. There are several items of note in the chapter. I particularly enjoyed learning how to use an old record player to construct a beveler capable of producing a nice 1–2 mm tip. But then, what will I do with my old Pink Floyd records? Preface ix
- 13. For precision, the microinjection needles need to include a mechanism for controlling the rate of injection. Two methods are given in Chapter 11 to achieve this: (1) using mercury to backfill the micropipette and relying upon the mercury to transduce and control pressure and (2) constructing a microneedle with a con- striction near the tip which reduces fluid movement and provides fine control of the injection. Both types of microinjection needles can be used in the same overall system. A similar microinjection system is described in Chapter 12, with modifications that allow for injection of the mouse oocyte within the complete follicle. Because the mouse oocyte exists within the follicle when in the ovary, it is intimately associated with the surrounding follicle cells until after ovulation. This rela- tively new method allows for the microinjection to occur while the oocyte is cultured within the intact follicle under more physiolo- gically relevant conditions. By microinjecting the follicle-enclosed oocytes, this group has been able to discover the mechanism(s) that maintain the immature oocyte arrested in meiosis and also to begin exploring the signaling mechanisms that are responsible for the reinitiation of meiosis (27–29). The technique is quite revolu- tionary and the concept of maintaining the proper physiological environment, as much as possible, is one that is applicable to all situations. In this chapter, you will also learn what type of music is most enjoyed by mouse oocytes. In some cases, microinjection would be very useful but not considered because of the perceived difficulty of injecting suffi- cient numbers of cells for further analysis. Chapter 13 describes a pressure-based method of injecting zygotes of the sea urchin Paracentrotus lividus. Using this method, up to several hundred embryos can be injected in a single session. In addition to the microinjection method, this chapter also describes experimental methods for perturbing gene function by either (1) producing and microinjecting synthetic mRNA produced in vitro, or (2) preparing linear DNA amplified by PCR for direct microinjection into the embryo. The advantages and disadvantages of these two different methods are discussed. The final two chapters describe practical uses of the technique of microinjection. Methods for imaging human gametes and zygotes after intracytoplasmic sperm injection (ICSI) are pre- sented in Chapter 14. The techniques focus on the identification of cytoskeletal elements and the role these components play dur- ing fertilization. Procedures are given in great detail for the removal of follicle cells and the zona pellucida, and fixation pro- cedures optimized for fluorescence immunocytochemistry and for examination by conventional electron microscopy and ultrastruc- tural immunolocalization. Chapter 15 presents a method for somatic cell nuclear trans- fer (SCNT) in the mouse. This article directly addresses the fact x Preface
- 14. that cloning by SCNT has always been difficult and inefficient (30, 31). In the protocol described here, donor nuclei from cumulus cells are injected directly into mouse oocyte. While this has been accomplished before, the Kishigami and Wakayama protocol results in a twofold to fivefold improvement in embryo develop- mental rates by the inclusion of trichostatin A, a histone deacety- lase inhibitor. The TSA may ‘reprogram’ the somatic cell nuclei, making them more amenable to the early developmental program. Also significant in Chapter 15 is a description of the use of a piezo-actuated micromanipulator that allows the use of larger microinjection needle tips with less damage to the oocyte. 3. Conclusion The methods described in this book should allow any lab to incorporate the technique of microinjection into their experimen- tal repertoire. Whether DNA, RNA, or protein is the molecule of interest, microinjection provides a mean of studying function within the context of the living cell. The technology is remarkably accessible and relatively inexpensive, while the possibilities are virtually endless. Acknowledgments I thank Dr. Laurinda Jaffe of the University of Connecticut Health Center for introducing me to microinjection and other fun things; and to Dr. John Walker of the University of Hertford- shire for his patience and guidance during the development of this book. References 1. Barber, M. (1904) A new method of isolat- ing microorganisms. J. Kans. Med. Soc. 4, 489–494. 2. Barber, M.A. (1911) A technique for the inoculation of bacteria and other sub- stances into living cells. J. Infect. Dis. 8, 348–360. 3. Chabry, L. (1887) Contribution a l’embryo- logie normal et teratologique des Ascidiens simples. Jour. de l’Anat. et de Physiol. 25, 167. 4. Fischer, J-L. (1990) Experimental embryol- ogy in France. Int. J. Dev. Biol. 34, 11–23. 5. Barber, M.A. (1914) The pipette method in the isolation of single microorganisms and in the inoculation of substances into living cells. Philippine J. Sci. B. 9, 307–360. 6. Korzh, V. and Strhle, U. (2002) Marshall Barber and the century of microinjection: from cloning of bacteria to cloning of every- thing. Differentiation 70, 221–226. Preface xi
- 15. 7. Lane, C.D., Marbaix, G., and Gurdon, J.B. (1971) Rabbit haemoglobin synthesis in frog cells: the translation of reticulocyte 9S RNA in frog oocytes. J. Mol. Biol. 61, 73–91. 8. Melton, D.A. (1987) Translation of messen- ger RNA in injected frog oocytes. Meth. Enzymol. 152, 288–296. 9. Masui, Y., and Markert, C.L. (1971) Cyto- plasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zool. 177, 129–145. 10. Reynhout J.K. and Smith L.D. (1974) Stu- dies on the appearance and nature of a maturation-inducing factor in the cytoplasm of amphibian oocytes exposed to progester- one. Dev. Biol. 38, 394–400. 11. Gurdon, J.B. (1968) Changes in somatic cell nuclei inserted into growing and maturing amphibian oocytes. J. Embryol. Exp. Morphol. 20, 401–414. 12. Evans, T., Rosenthal, E.T., Youngblom, J., Distel, D., Hunt, T. (1983) Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage divi- sion. Cell 33, 389–396. 13. Nurse, P., Thuriaux, P., and Nasmyth, K. (1976) Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet. 146, 167–178. 14. Feramisco, J.R. (1979) Microinjection of fluorescently labeled a-actinin into living fibroblasts. Proc. Natl. Acad. Sci. USA. 76, 3967–3971. 15. Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G., Cormier, M.J. (1992) Pri- mary structure of the Aequorea victoria green- fluorescent protein. Gene. 111, 229–233. 16. Shimomura, O. (2005) The discovery of aequorin and green fluorescent protein. J. Microsc. 217, 1–15. 17. Hiramoto, Y. (1962) Microinjection of the live spermatozoa into sea urchin eggs. Exp. Cell Res. 27, 416–426. 18. Kiehart, D.P. (1982) Microinjection of echi- noderm eggs: apparatus and procedures. Methods Cell Biol. 25, 13–31. 19. Kishimoto, T. (1986) Microinjection and cytoplasmic transfer in starfish oocytes. Methods Cell Biol. 27, 379–394. 20. Jaffe, L.A., and Terasaki, M. (2004) Quanti- tative microinjection of oocytes, eggs, and embryos. Methods Cell Biol. 74, 219–242. 21. Saunders, C.M., Larman, M.G., Parrington, J., Cox, L.J., Royse, J., Blayney, L.M., Swann, K., and Lai F.A. (2002) PLC: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development. Devel- opment 129, 3533–3544. 22. Corey, D.R., and Adams, J.M. (2001) Mor- pholino antisense oligonucleotides: tools for investigating vertebrate development. Gen- ome Biol. 2(5), reviews1015.1–1015.3. 23. Wu, C. and Muslin, A.J. (2002) Role of 14- 3-3 proteins in early Xenopus development. Mech. Dev. 119, 45–54. 24. Yergeau, D.A., and Mead, P.E. (2007) Manipulating the Xenopus genome with transposable elements. Genome Biol. 8 (Suppl 1), S11. 25. Pan, F.C., Chen, Y., Loeber, J., Henning- feld, K., Pieler, T. (2006) I-SceI meganu- clease-mediated transgenesis in Xenopus. Dev Dyn. 235, 247–252. 26. Etkin, L., Pearman, B., Roberts, M., Bektesh, S.L. (1984) Replication, integra- tion and expression of exogenous DNA injected into fertilized eggs of Xenopus laevis. Differentiation 26, 194–202. 27. Mehlmann, L.M., Jones, T.L., Jaffe, L.A. (2002) Meiotic arrest in the mouse follicle maintained by a Gs protein in the oocyte. Science 297, 1343–1345. 28. Mehlmann, L.M., Saeki, Y., Tanaka, S., Brennan, T.J., Evsikov, A.V., Pendola, F.L., Knowles, B.B., Eppig, J.J., Jaffe, L.A. (2004) The Gs-linked receptor GPR3 maintains meiotic arrest in mammalian oocytes. Science 306, 1947–1950. 29. Freudzon, L., Norris, R.P., Hand, A.R., Tanaka, S., Saeki, Y., Jones, T.L., Rasenick, M.M., Berlot, C.H., Mehlmann, L.M., Jaffe, L.A. (2005) Regulation of meiotic prophase arrest in mouse oocytes by GPR3, a consti- tutive activator of the Gs G protein. J. Cell Biol. 171, 255–265. 30. Tian, X.C., Kubota, C., Enright, B., Yang, X. (2003) Cloning animals by somatic cell nuclear transfer – biological factors. Reprod Biol Endocrinol. 1, 98. 31. Campbell, K.H., Fisher, P., Chen, W.C., Choi, I., Kelly, R.D., Lee, J.H., Xhu, J. (2007) Somatic cell nuclear transfer: Past, present and future perspectives. Theriogeno- logy. 68 Suppl 1, S214–S231. xii Preface
- 16. Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv 1 Expression of Exogenous mRNA in Xenopus laevis Embryos for the Study of Cell Cycle Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Jill C. Sible and Brian N. Wroble 2 Use of Luciferase Chimaera to Monitor PLC Expression in Mouse Eggs . . . . . . . . . 17 Karl Swann, Karen Campbell, Yuansong Yu, Christopher Saunders and F. Anthony Lai 3 Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino Oligos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Jeffrey M. C. Lau and Anthony J. Muslin 4 A Microinjectable Biological System, the Xenopus Oocyte, as an Approach to Understanding Signal Transduction Protein Function . . . . . . . . . . . . . . . . . . . . . . . . 43 Katia Cailliau and Edith Browaeys-Poly 5 Combining Microinjection and Immunoblotting to Analyze MAP Kinase Phosphorylation in Single Starfish Oocytes and Eggs . . . . . . . . . . . . . . . . . . . . . . . . . 57 David J. Carroll and Wei Hua 6 Analysis of Signaling Pathways in Zebrafish Development by Microinjection . . . . . . . 67 William H. Kinsey 7 Protein Inhibition by Microinjection and RNA-Mediated Interference in Tissue Culture Cells: Complementary Approaches to Study Protein Function . . . . . . . . . . . . 77 Jane R. Stout, Rania S. Rizk, and Claire E. Walczak 8 DNA Delivery by Microinjection for the Generation of Recombinant Mammalian Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Sebastien Chenuet, Madiha Derouazi, David Hacker and Florian Wurm 9 Bacteriophage fC31 Integrase Mediated Transgenesis in Xenopus laevis for Protein Expression at Endogenous Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Bryan G. Allen and Daniel L. Weeks 10 Germline Transformation of Caenorhabditis elegans by Injection . . . . . . . . . . . . . . . 123 Pavan Kadandale, Indrani Chatterjee and Andrew Singson 11 Quantitative Microinjection of Mouse Oocytes and Eggs . . . . . . . . . . . . . . . . . . . . . 135 Douglas Kline 12 Microinjection of Follicle-Enclosed Mouse Oocytes . . . . . . . . . . . . . . . . . . . . . . . . . 157 Laurinda A. Jaffe, Rachael P. Norris, Marina Freudzon, William J. Ratzan, and Lisa M. Mehlmann 13 Functional Studies of Regulatory Genes in the Sea Urchin Embryo . . . . . . . . . . . . . 175 Vincenzo Cavalieri, Maria Di Bernardo, and Giovanni Spinelli xiii
- 17. 14 Exploring the Cytoskeleton During Intracytoplasmic Sperm Injection in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Vanesa Y. Rawe and Héctor Chemes 15 Somatic Cell Nuclear Transfer in the Mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 Satoshi Kishigami and Teruhiko Wakayama Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 xiv Contents
- 18. Contributors BRYAN G. ALLEN Department of Biochemistry, University of Iowa, Iowa City, IA, USA EDITH BROWAEYS-POLY Université des Sciences et Technologies de Lille, Laboratoire de Régulation des Signaux de Division, Villeneuve d’Ascq Cedex, France KATIA CAILLIAU Université des Sciences et Technologies de Lille, Laboratoire de Régulation des Signaux de Division, Villeneuve d’Ascq Cedex, France KAREN CAMPBELL Department of Obstetrics and Gynaecology, School of Medicine, Cardiff University, Cardiff, UK DAVID J. CARROLL Department of Biological Sciences, Florida Institute of Technology, Melbourne, FL, USA VINCENZO CAVALIERI Dipartimento di Biologia Cellulare e dello Sviluppo ‘‘A. Monroy’’, Università di Palermo, Palermo, Italy INDRANI CHATTERJEE Waksman Institute, Rutgers University, Piscataway, NJ, USA HÉCTOR CHEMES CEDIE, Laboratorio de Fisiologı́ y Patologı́a Testicular, Hospital de Niños ‘Ricardo Gutiérrez’, Buenos Aires, Argentina SEBASTIEN CHENUET École Polytechnique Féderale de Lausanne, EPFL-SV-IBI-LBTC, Lausanne, Switzerland MADIHA DEROUAZI École Polytechnique Féderale de Lausanne, EPFL-SV-IBI-LBTC, Lausanne, Switzerland MARIA DI BERNARDO Istituto di Biomedicina e Immunologia Molecolare ‘‘A. Monroy’’, Consiglio Nazionale delle Ricerche, Palermo, Italy MARINA FREUDZON Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA DAVID HACKER École Polytechnique Féderale de Lausanne, EPFL-SV-IBI-LBTC, Lausanne, Switzerland WEI HUA College of Aqua-life Science and Technology, Shanghai Fisheries University, Shanghai, China LAURINDA A. JAFFE Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA PAVAN KADANDALE Waksman Institute, Rutgers University, Piscataway, NJ, USA WILLIAM H. KINSEY Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS, USA SATOSHI KISHIGAMI RIKEN, Center for Developmental Biology, Kobe, Japan DOUGLAS KLINE Department of Biological Sciences, Kent State University, Kent, OH, USA F. ANTHONY LAI Cell Signaling Laboratory, Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff, UK JEFFREY M.C. LAU Center for Cardiovascular Research, Department of Medicine, Department of Cell Biology Physiology, Washington University School of Medicine, St. Louis, MO, USA xv
- 19. LISA M. MEHLMANN Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA ANTHONY J. MUSLIN Center for Cardiovascular Research, Department of Medicine, Department of Cell Biology Physiology, Washington University School of Medicine, St. Louis, MO, USA RACHAEL P. NORRIS Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA WILLIAM J. RATZAN Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA VANESA Y. RAWE Centro de Estudios en Ginecologı́a y Reproducción (CEGyR), Buenos Aires, Argentina RANIA S. RIZK Department of Biology, Indiana University, Bloomington, IN, USA CHRISTOPHER SAUNDERS Cell Signaling Laboratory, Wales Heart Research Institute, School of Medicine, Cardiff University, Cardiff, UK JILL C. SIBLE Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA ANDREW SINGSON Waksman Institute, Rutgers University, Piscataway, NJ, USA GIOVANNI SPINELLI Dipartimento di Biologia Cellulare e dello Sviluppo ‘‘A. Monroy’’, Università di Palermo, Palermo, Italy JANE R. STOUT Department of Biochemistry and Molecular Biology, Indiana University Medical Sciences, Bloomington, IN, USA KARL SWANN Department of Obstetrics and Gynaecology, School of Medicine, Cardiff University, Cardiff, UK TERUHIKO WAKAYAMA RIKEN, Center for Developmental Biology, Kobe, Japan CLAIRE E. WALCZAK Department of Biochemistry and Molecular Biology, Indiana University Medical Sciences, Bloomington, IN, USA DANIEL L. WEEKS Department of Biochemistry, University of Iowa, Iowa City, IA, USA BRIAN N. WROBLE Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA FLORIAN WURM École Polytechnique Féderale de Lausanne, EPFL-SV-IBI-LBTC, Lausanne, Switzerland YUANSONG YU Department of Obstetrics and Gynaecology, School of Medicine, Cardiff University, Cardiff, UK xvi Contributors
- 20. Chapter 1 Expression of Exogenous mRNA in Xenopus laevis Embryos for the Study of Cell Cycle Regulation Jill C. Sible and Brian N. Wroble Abstract The microinjection of mRNA that is transcribed and capped in vitro into fertilized eggs and embryos of Xenopus laevis provides a powerful means for discovering the function of proteins during early develop- ment. Proteins may be overexpressed for a gain-of-function effect or exogenous protein function may be compromised by the microinjection of mRNA encoding ‘‘dominant-negative’’ proteins. This methodol- ogy is particularly suited for the investigation of the regulation of the cell cycle, checkpoints, and apoptosis in early development. Key words: Microinjection, mRNA, cell cycle, Xenopus laevis, early development, apoptosis, checkpoints, embryos, in vitro transcription. 1. Introduction 1.1. Applications The microinjection of mRNA transcribed in vitro into oocytes, fertilized eggs, and embryonic cells from Xenopus laevis has become a classic methodology in developmental biology that takes advantage of the highly efficient translational capability of these cells. RNAs encoding membrane channels and transporters (other species) can be expressed in oocytes and eggs, providing a large surface area for patch clamping and other electrophysiologi- cal studies (1–3). Microinjection of mRNAs encoding X. laevis proteins enables one to determine the effect of ectopic expression of endogenous genes on early development. mRNAs of genes that have been mutated to encode ‘‘dominant-negative’’ proteins can be microinjected into embryos to assess the developmental David J. Carroll (ed.), Microinjection: Methods and Applications, Vol. 518 Ó 2009 Humana Press, a part of Springer ScienceþBusiness Media, LLC DOI 10.1007/978-1-59745-202-1_1 1
- 21. consequences of interfering with the function of a protein or pathway (4,5). Microinjection of mRNAs into X. laevis embryos provides a particularly powerful method for the investigation of cell cycle regulation. The first twelve cell cycles are driven exclusively by the translation of maternal cyclin mRNAs (6), and thus are amenable to manipulation by exogenous mRNA. After the twelfth cell cycle, the midblastula transition (MBT) begins, and cell cycle becomes lengthened and asynchronous (7). The embryo also becomes transcriptionally active at the MBT (8). Many maternal mRNAs that regulate the cell cycle are degraded and replaced by zygotic isoforms (9–11). Exogenous mRNAs encoding cell cycle-related proteins often alter the timing of cleavage cycles, the onset of the MBT, or both. The MBT also delineates a remodeling of the cell cycle with respect to cell cycle checkpoints. Prior to the MBT, embryos do not arrest cleavage divisions in response to damaged or unreplicated DNA, but rather undergo a maternally regulated program of apoptosis during early gastrulation (12–14). After the MBT, cell cycle checkpoints become operational, and damaged or unreplicated DNA triggers cell cycle arrest rather than apoptosis. Thus, the early embryo of X. laevis provides a dynamic system of cell cycle remodeling (15) that can be readily manipulated by microinjection of mRNA transcribed in vitro. 1.2. Relationship to Other Methods to Manipulate Gene Expression in X. laevis In recent years, the suite of tools to manipulate gene expression in X. laevis and other externally developing organisms has been enhanced with new technologies. Notably, the development of transgenesis methodologies for X. laevis (16) and X. tropicalis (17) enables researchers to introduce genes under the control of specific promoters stably into the genome. Antisense morpholi- nos can be microinjected into the embryo to block translation of specific mRNAs through the late embryonic stages (18). None- theless, the more classic methodology of expressing genes by the microinjection of mRNAs transcribed in vitro maintains its value in the study of early embryogenesis. Because X. laevis embryos do not transcribe their genome until the MBT (cell cycle 12, approxi- mately 6 h post-fertilization), transgenes will not be expressed before the MBT. Therefore, early developmental events including cell cycle remodeling and activation of the maternal program of apoptosis will not be affected by transgenesis, but they can be altered by microinjection of mRNAs, which are efficiently trans- lated in the early embryo. Antisense morpholinos will block trans- lation of maternal mRNAs but will not affect maternally supplied proteins, many of which regulate the cell cycle and apoptosis. In addition to being particularly suited for manipulation of gene expression during the earliest stages of development, microinjec- tion of mRNAs is a relatively simple and inexpensive technique. 2 Sible and Wroble
- 22. 1.3. Summary Investigation of the regulation of cell cycle remodeling and other early developmental events can be facilitated by the microinjection of mRNA into early X. laevis embryos. Although the methodol- ogies are straightforward, care should be taken to design plasmid constructions that encode protein tags and a poly-A tail, to gen- erate high-quality RNA in an RNase-free environment, and to control for nonspecific effects of exogenous mRNA. By pairing this methodology with biochemical and morphological assays for cell cycle progression and apoptosis, we continue to build a sys- tems-level view of the signaling networks that control early devel- opmental events. 2. Materials 2.1. Template 1. pSP64poly(A) plasmid (Promega Corp., Madison, WI, USA) (see Note 1). 2. Restriction enzymes such as EcoRI and PvuII (see Note 2). 3. RNase-free phenol/chloroform (1:1). 4. 3-M sodium acetate. 5. 70% and 100% ethanol. 6. Tris-EDTA (TE) buffer: 10-mM Tris, 1-mM EDTA, pH 8.0. 2.2. RNA Preparation 1. mMessage mMachineTM (Ambion/Applied Biosystems, Inc., Austin, TX, USA). 2. Isopropanol. 3. Phenol: chloroform (1:1) 2.3. Fertilizing Eggs for Microinjection 1. Pregnant mare serum gonadotropin (PMSG; Calbiochem, catalog #367222). 2. Human chorionic gonadotropin (HCG; Sigma, catalog #C1063). 3. 0.1X MMR (0.5-mM HEPES, pH 7.8, 10-mM NaCl, 0.2-mM KCl, 0.1-mM MgSO4, 0.2-mM CaCl2, 0.01-mM EDTA). 2.4. Injecting the mRNA 1. Microcapillary tubes for making the microinjection needles. 2. Micropipet puller such as a Narishige PC-10 (Narishige Scien- tific Instrument Lab, Inc., Tokyo, Japan). 3. Sexually mature female adult Xenopus laevis. Commercial sup- pliers include (1) Xenopus I, Inc., Dexter, MI, USA; or (2) Xenopus Express Inc., Brooksville, FL, USA. Expression of Exogenous mRNA in Xenopus 3
- 23. 2.5. Monitoring Expression of the mRNA 1. Mouse M2 Anti-FLAG monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA). 3. Methods 3.1. Preparing the Template 3.1.1. Constructing/ Selecting the Plasmid Vector Encoding the Experimental and Control mRNAs Several considerations in the design and construction of the plas- mid template should be made to optimize and verify the translat- ability of the mRNA to be injected. 1. The vector should have a bacteriophage T7, T3, or SP6 pro- moter upstream of a multicloning site (MCS). The pSP64po- ly(A) plasmid (Promega, catalog #P1241) is recommended because it encodes a poly-A tail downstream of the MCS followed by a unique restriction site (See Note 1). 2. The cDNA to be inserted into the MCS can be generated by excision from an existing plasmid followed by ligation into the in vitro transcription vector if compatible restriction sites exist. Alternatively, the desired portion of the cDNA (with or with- out UTRs) can be amplified by PCR. An advantage of PCR is that restriction sites and short epitope tags can be incorporated directly into the primers. 3. As an example of this strategy, the primer sequences and PCR conditions for generating a FLAG-tagged Chk2 cDNA to be cloned into pSP64polyA are shown in Fig. 1.1. A two-stage PCR was used because the 50 ends of each primer containing FLAG sequence and restriction sites will not anneal to the original cDNA template, necessitating a lower annealing temperature. After several rounds of amplification, enough product incorporating these primers is available as template and the annealing temperature can be raised to allow for a more effi- cient amplification. 4. The PCR product can then be digested with the appropriate restriction enzymes and cloned into digested pSP64polyA vector. Once clones with inserts have been selected, amplified, and sequenced, large-scale preparations of the plasmid should be made. 3.1.2. Restriction Digestion of the Template 1. Linearize 30-mg template plasmid by restriction digestion with an appropriate enzyme. 2. The restriction enzyme should not generate 30 overhangs. Try to choose a site that generates 50 overhangs (best choice) or blunt ends (OK). 3. A typical digestion reaction would include: a. 30-mg plasmid template b. 10-ml 10X buffer for restriction enzyme 4 Sible and Wroble
- 24. c. 2-ml restriction enzyme d. H2O to 100-ml total volume 4. Incubate at 37°C for 5 h to overnight. 5. Digestion should be to completion because undigested plas- mid can serve as template for transcription of concatamers of the entire plasmid. 6. To test the reaction for completion of digestion, remove 1 ml of the reaction and resolve by agarose gel electrophoresis alongside the same concentration of uncut plasmid. Linearized plasmid should resolve as a single discrete band that typically migrates slower than uncut plasmid. 7. After digestion, heat the reaction mixture at 65°C for 15 min to inactivate it. Extract with an equal volume of phenol/ chloroform (RNase-free). 8. From this point on, use only RNase-free reagents and plastic- ware and practice good RNA technique. 9. Precipitate the DNA with 1/10 volume 3-M sodium acetate and 2 volumes ethanol (EtOH) at –20°C overnight or –80°C for at least 15 min. 10. Centrifuge the DNA at 16,000 g for 15–30 min at 4°C, remove the 100% EtOH, wash the pellet with 70% EtOH (for RNA), centrifuge, remove the EtOH and air-dry the pellet. 1 cycle @ 94°C for 2 min 5 cycles @ 94°C for 30 sec 52°C for 45 sec 68°C for 6 min 1 cycle @ 68°C for 20 min 20 cycles @ 94°C for 30 sec 62°C for 45 sec 68°C for 6 min 1 cycle @ 68°C for 20 min 1 7 2 3 4 5 6 8 9 A B Fig 1.1. PCR strategy for cloning X. laevis Chk2 cDNA into pSP64polyA. (A) Forward and reverse primers. On forward primer: 1, extra bases to facilitate restriction digestion of PCR product; 2, PstI site; 3, start codon; 4, Flag tag; 5, Chk2-specific sequence beginning after the start codon. On reverse primer: 6, extra bases to facilitate restriction digestion of PCR product, BamHI site; 8, reverse complement of stop codon; 9, reverse complement of the end of the Chk2 ORF. (B) Two-stage PCR program for amplifying Chk2 from cDNA clone using above primers. Expression of Exogenous mRNA in Xenopus 5
- 25. 11. Resuspend in 30-mL TE (for RNA). 12. Take1mlanddeterminetheconcentrationbyspectrophotometry. 13. Dilute the DNA to 1 mg/ml. Store unused portion at 4°C or –20°C. This is your template DNA for in vitro transcription. 3.2. In Vitro Transcription of the mRNA The recommended reagents for the in vitro transcription are sup- plied by Ambion (www.ambion.com) in the mMessage mMachi- neTM kit. Following the manufacturer’s instructions will generate sufficient amounts of mRNA suitable for microinjection. The pro- tocol described below has been modified slightly to increase yields. 1. Set up the following reaction using your template DNA and reagents from the mMessage mMachine kit: a. 4-ml RNase-free water (there is some in the kit) b. 2-ml 10X transcription buffer (be sure this is completely thawed and there is not a precipitate in the tube) c. 10-ml 2X ribonucleotide mix d. 2-ml template DNA (0.5 mg/ml) e. 2-ml SP6, T3, or T7 polymerase (be sure you are using the right one) 2. Incubate at 37°C for 1 h. 3. Add 1 ml DNase I, mix well and incubate at 37°C for 15 min. 4. Add 115 ml RNase-free water and 15-ml NH4OAc (in kit). 5. Extract with 150-ml phenol/chloroform. 6. Add 150-ml isopropanol and precipitate at –20°C for a few hours to overnight. 7. Centrifuge at 16,000 g for 20 min at 4°C, wash with 70% EtOH (RNase-free) as described above, dry the pellet briefly and resuspend in 20 ml RNase-free water or TE. 8. Take1mlanddeterminetheconcentrationbyspectrophotometry. 9. Take 1 ml and resolve by denaturing gel electrophoresis to confirm that the RNA is intact and of the appropriate size. An example is shown in Fig. 1.2. 10. Store the remaining RNA at –80°C in several aliquots (see Note 3). 3.3. Fertilizing Eggs for Microinjection 1. To induce egg-laying, inject female X. laevis subcutaneously into the dorsal lymph sac with 75 IU PMSG (Calbiochem, catalog #367222) 3–5 days before eggs are desired. 2. Approximately 12–16 h before eggs are needed, inject the frogs with 550 IU HCG (Sigma, catalog #C1063). 3. The next day, collect freshly laid eggs directly into a petri dish containing 0.1X MMR (0.5-mM HEPES, pH 7.8, 6 Sible and Wroble
- 26. 10-mM NaCl, 0.2-mM KCl, 0.1-mM MgSO4, 0.2-mM CaCl2, 0.01-mM EDTA). 4. Allow fertilization to proceed undisturbed for 10 min, then dejelly eggs in freshly prepared solution of 2% cysteine in 0.1X MMR (see Note 4). 5. Dejellying will take approximately 5–7 min and is complete when eggs pack tightly and geometrically together. As soon as eggs are dejellied, wash eggs 4–6 times in 0.1X MMR. 6. Examine eggs under the microscope. In fertilized eggs, the ani- mal pole will be slightly contracted (occupying less than a full hemisphere of the egg) and will be oriented upward (Fig 1.3A). Fertilized eggs are typically firm when prodded with forceps, like a full balloon. Unfertilized eggs are soft, randomly oriented, and will often stick to the surface of the petri dish (Fig. 1.3B). Sometimes fertilized eggs will remain soft. These are viable but much harder to microinject (Fig. 1.3H). 7. Fertilized eggs should be left undisturbed for 30 min after fertilization before they are microinjected. This allows time for cortical rotation, which establishes the dorsal–ventral axis. 3.4. Injecting the mRNA 3.4.1. Preparing Needles for Microinjection 1. To inactivate contaminating RNases, the microcapillary tubes used to make the microinjection needles should be baked at 80°C for 2 h before they are pulled into needles. 2. The needles can then be pulled using a standard micropipet puller (e.g., Narishige PC-10). 3. Pulled needles should then be attached to the microinjector/ micromanipulator and calibrated using RNase-free water or TE. The tip of the needle typically needs to be broken with a fine, sharp pair of forceps (Fig. 1.3C). 4. The needle should be calibrated to inject the desired amount of RNA (50 ng) in the desired volume (50 nl). 2.3 kb 1.3 kb ~1.6 kb MW Chk2 mRNA Fig. 1.2. Ethidium-bromide-stained product of in vitro transcription reaction. RNA was resolved on 1% MOPS agarose gel and visualized under UV light. MW ¼ molecular weight marker. Expression of Exogenous mRNA in Xenopus 7
- 27. Fig. 1.3. Stereoscopic images of eggs and the microinjection procedure. (A) Fertilized eggs from X. laevis. Note the contraction of the pigments in the animal hemisphere. (B) A clutch with unfertilized eggs. Animal hemisphere is not contracted and egg remains soft. (C) The tip of a microinjection needle being broken with forceps. (D) The diameter of drops of the injection solution onto parafilm is used to estimate the volume. Note: micrometer is not visible because it is located in the eyepiece of the microscope. (E) Injecting a one-celled embryo. (F) Injecting a two-celled embryo. (G) Injecting a four-celled embryo. (H) Injecting a soft embryo. Embryo is fertilized but soft and therefore challenging to inject without breaking the needle or pushing the needle in too far. Scale bars ¼ 1 mm. 8 Sible and Wroble
- 28. An easy way to calibrate is to expel a drop of the injection solution onto a piece of parafilm and measure the radius (r) of the drop with a micrometer (Fig. 1.3D). The volume (V) of the drop can then be determined (V ¼ 4/3pr3 ). 5. The injection volume should then be adjusted (by manipulat- ing injection pressure or time with an automated microinjector or volume directly with a manual microinjector) to achieve the desired volume (see Note 5). 3.4.2. Performing Injections 1. For injection at the one-cell stage (Fig. 1.3E), embryos can be injected from 30 min post-fertilization (pf) until signs that the first cleavage is beginning, usually 90 min pf, but some- times as early as 60 min pf in warmer ambient temperatures. During this 30–60 min window, an experienced investigator can inject several hundred embryos with 2–5 different mRNAs. 2. Embryos should be injected near the interface of the animal and vegetal hemispheres with care to avoid the animal half where the nucleus resides. Most investigators find it easiest to position the needle so that it will inject in the proper place then bring each embryo to and from the needle with a pair of blunt forceps. 3. The embryos should be pushed onto the needle until it just breaches the membrane. For firm eggs, this will feel like insert- ing a needle into a full balloon (Figs. 1.3E–G). For very soft embryos, injections will be much more difficult, like piercing a very soft, understuffed pillow (Fig. 1.3H). 4. Depending on the experimental design, the investigator may wait to microinject mRNA at the two-cell stage (Fig. 1.3F). Injection of one of the two blastomeres leaves the remaining uninjected blastomere as an internal negative control. Some investigators inject both blastomeres at the two-cell stage because they have observed better viability than injections at the one-cell stage. 5. Likewise, one can inject a single blastomere at the 4- (Fig. 1.3G), 8-, 16-cell stage and so on. In these experiments, the investigator is typically targeting a particular blastomere of a specific cell line- age (19). These cell cycles last approximately 30 min each, and thus, the window of time for microinjection is shorter. 3.5. Monitoring Expression of the mRNA 1. Expression of the microinjected mRNA should be verified by western blotting of embryo extracts. 2. If the mRNA encodes an epitope tag, such as the FLAG tag described in Section 3.1.1, then blotting for that epitope can be performed with a commercially available antibody (Fig. 1.4A). 3. For the FLAG tag, the mouse M2 monoclonal anti-FLAG antibody works well although there is recognition of a Expression of Exogenous mRNA in Xenopus 9
- 29. nonspecific band at approximately 37 kDa. Blotting for an epitope tag allows the same antibody to be used to verify translation of all mRNAs including the control mRNA. 4. If the mRNA does not encode a tagged protein, then an anti- body specific for the encoded protein may be used instead. In this case, endogenous protein will also be detected, and there- fore, comparison of relative amounts of endogenous to exo- genous protein can be made by comparing western blotting analysis of experimental embryos to uninjected or control- injected embryos. This information is valuable for estimating the amount of dominant-negative exogenous protein relative to the endogenous, wild-type protein. 3.6. Assaying the Effects of the mRNA on Embryonic Development 3.6.1. Monitoring Embryonic Morphology 1. When using microinjection of mRNA to investigate cell cycle regulation and apoptosis in early embryos, phenotypic changes may manifest early in development, and thus, embryos should be monitored closely for the first 12 h of life (see Note 6). 2. Expression of some mRNAs will induce a modest delay in cleavage time, a delay that may not be appreciable for several cell cycles, but can be discriminated based on number and size of cells at the midblastula stage. 3. Examples of mRNAs that modestly delay cleavage include those encoding: 34-Xic1, an inhibitor of cyclin E-Cdk2 kinase (20); low doses of the cell cycle checkpoint kinase Chk2 (Fig. 1.4B) (5); and the Cdk inhibitory kinase Wee2 (11). 4. Likewise, mRNAs encoding cell cycle activators may accelerate cleavage cycles. Examples include cyclin B mRNA (21) and the Cdk-activating phosphatase Cdc25A (22). 3.6.2. Assays for Cell Cycle Progression In addition to monitoring cleavage cycles by gross morphology, assays for biochemical and nuclear changes in the cell cycle are summarized here with references to articles providing examples and protocols. 1. Cell cycles can be monitored biochemically by western blotting for the mitotic cyclins or enzymatic assays for Cdk activity (20,21). For these experiments, approximately five embryos per time-point should be collected and snap-frozen. Time-points should be collected every 5–10 min in order to detect oscillations. Only well-synchronized clutches of embryos should be used in order to detect clear mitotic peaks. 2. To monitor cell cycle progression via rounds of DNA repli- cation, incorporation of 3 H thymidine into DNA can be followed (13). 10 Sible and Wroble
- 30. These assays provide a different perspective of the cell cycle since cleavages will occur even in the absence of DNA replica- tion in pre-MBT embryos. 3.6.3. Assays for the Midblastula Transition Expression of mRNAs that alter the rate of cleavage cycles will also affect the timing of most events of the MBT; however, some events, such as the degradation of maternal cyclin E, do not depend on reaching a critical nucleo-cytoplasmic ratio (20). Therefore, assays that monitor several hallmarks of the MBT Fig 1.4. Assessment of the effect of microinjection. (A) Western blot for the FLAG epitope on the Chk2 protein. Each lane contains the equivalent of one embryo although extracts from five embryos were pooled for each sample. Note that embryos with low levels of FLAG-tagged protein did not show a cell cycle delay phenotype. (B) Cell cycle delay in embryos injected with Chk2 mRNA versus luciferase (mRNA). Delay is apparent because the cells from embryos injected with Chk2 are larger and fewer. (C) Embryo undergoing apoptosis. Arrows show the area where cells have detached and are filling the space delineated by the vitelline membrane. Scale bars ¼ 1 mm. Expression of Exogenous mRNA in Xenopus 11
- 31. should be employed to best understand the effect of the exogen- ous mRNA on early development. 1. The onset of zygotic transcription is a classic definition of the MBT and occurs at a critical nucleo-cytoplasmic ratio. Transcription can be monitored by northern blotting for expression of an early developmental gene such as GS17 (13). 2. Alternatively, a more global picture of transcription can be obtained by loading embryos with 3 H uridine and then follow- ing incorporation of 3 H into RNA (13). 3. At the MBT, a host of changes to cell cycle proteins occurs, as cell cycles lengthen and come under control of the zygotic genome. Cell cycle changes that can be assayed by western blot analysis include degradation of maternal cyclin E (20), Cdc25A (22), and Wee1 (23) as well as increased tyrosine phosphorylation of Cdk1 and Cdk2 (5,22). 3.6.4. Assays for Apoptosis Many exogenous mRNAs trigger a maternally regulated program of apoptosis in early X. laevis embryos. Induction of apoptosis can be readily distinguished from a nonspecific toxic effect of an mRNA. Apoptosis will not be initiated until the early gastrula stage and is characterized by striking morphological events (Fig. 1.4C). 1. In apoptotic embryos, cell lose their attachments from one another and dissociated cells come ‘‘bursting’’ from the blas- tocoel, eventually filling up the cavity enclosed by the vitelline membrane. A clutch of embryos will undergo apoptosis with good synchrony, with the entire clutch showing an apoptotic phenotype within an hour. 2. Although this apoptotic morphology is distinct to the trained eye, apoptosis should be verified by one or more specific assays. The condensation of chromatin during apoptosis can be iden- tified by electron microscopy or fluorescence microscopy of sectioned embryos stained with a dye that binds DNA (13). However, not every nucleus in the embryo may appear apoptotic and some agents that induce apoptosis, such as those that block DNA replication, do not result in typical apoptotic bodies (4). 3. The detection of DNA ladders by agarose gel electrophoresis is another common assay for apoptosis. These ladders can be detected in DNA isolated from apoptotic X. laevis embryos (12), but the resolution is often poor, obscured by abundant RNA, despite extensive treatment with RNases. 4. Assays that have proven more reliable in detecting apoptosis in X. laevis embryos are the whole-mount TUNEL assay and poly-(ADP ribose) polymerase (PARP) cleavage assay. The TUNEL assay is based on a modification of the whole-mount in situ hybridization protocol for X. laevis embryos (13,14). 12 Sible and Wroble
- 32. Embryos are fixed, and incubated with terminal deoxy- transferase (TdT) and digoxigenin-labeled dUTP (dig- dUTP). TdT catalyzes the addition of dig-dUTP to free 30 - OH ends of DNA An alkaline phosphatase conjugated anti-dig antibody and chromagens are used to detect the labeled nuclei. Because the chromagenic precipitate is a dark purple color that can be obscured by pigment, embryos generated from albino females are typically used. 5. In the PARP assay, embryos are collected when there is morphologic indication of apoptosis. Embryos are lysed and lysates are incubated with recom- binant PARP which is then analyzed by western blotting (4). Detection of an 85-kDa cleavage fragment of PARP indicates activation of the apoptotic effector enzyme caspase 3. Because recombinant human PARP is effectively cleaved by apoptotic extracts from X. laevis embryos, all necessary reagents are commercially available. 4. Notes 1. The poly-A tail is thought to improve translatability and/or stability of the mRNA. However translatable mRNAs can be synthesized in vitro using templates that do not encode a poly- A tail. Inclusion of 50 and 30 untranslated sequences from the gene may also improve efficiency of translation of the mRNA, but generally is not necessary. 2. For pSP64poly A, EcoRI is the preferred restriction endo- nuclease because it cuts just after the poly-A tail. If your cDNA contains an EcoRI restriction site, then PvuII is a good alternative. It cuts 182 bp downstream of the poly-A tail. 3. To improve yields and efficiency of recovery even further, set up duplicate or triplicate reactions, pool prior to phe- nol/chloroform extraction, and complete the procedure, adjusting volumes accordingly. In practice, yields are greater than 2–3 times that of a single reaction, probably due to more efficient recovery in the extraction and pre- cipitation steps. 4. Prolonged exposure to cysteine will damage the eggs. 5. Some investigators find it useful to calibrate several needles ahead of time, but the calibration should be rechecked just Expression of Exogenous mRNA in Xenopus 13
- 33. prior to injecting the embryos. Experienced investigators typi- cally calibrate needles ‘‘on-the-fly’’ as they are needed. 6. Physical penetration of an egg and expression of exogenous RNA could introduce artifacts that affect development. For example, cleavage cycles may lengthen if exogenous mRNA competes with cyclin mRNA for access to the translational machinery. Therefore, control embryos should be microin- jected with mRNA encoding an inert or irrelevant protein. A FLAG-tagged luciferase cDNA cloned into pSP64poly A is one useful vector for making control mRNA (24). Alternatively, a cDNA clone encoding the protein of interest could be muta- genized to be catalytically inactive or otherwise inert. References 1. Kinoshita-Kawada M., Oberdick J., and Xi Zhu M. (2004) A Purkinje cell specific GoLoco domain protein, L7/Pcp-2, modu- lates receptor-mediated inhibition of Cav2.1 Ca2+ channels in a dose-dependent manner. Brain Res. 132, 73–86. 2. Brandt S. and Fisahn J. (1998) Identification of a K+ channel from potato leaves by func- tional expression in Xenopus oocytes. Plant Cell Physiol. 39, 600–6. 3. Morales M.M., Carroll T.P., Morita T., Schwiebert, E.M., Devuyst O, Wilson P.D., Lopes A G., Stanton B.A., Dietz H.C., Cut- ting G.R., and Guggino W.B. (1996) Both the wild type and a functional isoform of CFTR are expressed in kidney. Am. J. Physiol. 270, F1038–48. 4. Carter A., and Sible J. (2003) Loss of XChk1 function leads to apoptosis after the midblas- tula transition in Xenopus laevis embryos. Mech. Devel. 120, 315–23. 5. Wroble B., Sible J. (2005) Chk2/Cds1 pro- tein kinase blocks apoptosis during early development of Xenopus laevis. Dev. Dyn. 233, 1359–65. 6. Murray A.W., and Kirschner M.W. (1989) Cyclin synthesis drives the early embryonic cell cycle. Nature 339, 275–80. 7. Newport J. and Kirschner M. (1982) A major developmental transition in early Xenopus embryos: I. Characterization and timing of cellular changes at the midblastula stage. Cell 30, 675–86. 8. Newport J. and Kirschner M. (1982) A major developmental transition in early Xenopus embryos: II. Control of the onset of tran- scription. Cell 30, 687–96. 9. Howe J.A., Howell M., Hunt T., Newport J.W. (1995) Identification of a developmen- tal timer regulating the stability of embryonic cyclin A and a new somatic A-type cyclin at gastrulation. Genes Dev. 9, 1164–76. 10. Howe J.A., and Newport J.W. (1996) A developmental timer regulates degradation of cyclin E1 at the midblastula transition during Xenopus embryogenesis. Proc. Natl. Acad. Sci. USA 93, 2060–4. 11. Leise W.F., III and Mueller P.R. (2002) Multiple Cdk1 inhibitory kinases regulate the cell cycle during development. Dev. Biol. 249, 156–73. 12. Anderson J.A., Lewellyn A.L., and Maller J.L. (1997) Ionizing radiation induces apop- tosis and elevates cyclin A1-Cdk2 activity prior to but not after the midblastula transi- tion in Xenopus. Mol. Biol. Cell 8, 1195–206. 13. Sible J.C., Anderson J.A., Lewellyn A.L., and Maller J.L. (1997) Zygotic transcription is required to block a maternal program of apoptosis in Xenopus embryos. Dev. Biol. 189, 335–46. 14. Hensey C., and Gautier J. (1997) A develop- mental timer that regulates apoptosis at the onset of gastrulation. Mech. Devel. 69, 183–95. 15. Frederick D.L., Andrews M.T. (1994) Cell cycle remodeling requires cell–cell interac- tions in developing Xenopus embryos. J. Exp. Zool. 270, 410–6. 16. KrollK.L.,AmayaE.(1996)TransgenicXenopus embryos from sperm nuclear transplantations 14 Sible and Wroble
- 34. reveal FGF signaling requirements during gas- trulation. Development 122, 3173–83. 17. Offield M.F., Hirsch N., and Grainger R.M. (2000) The development of Xenopus tropi- calis transgenic lines and their use in studying lens developmental timing in living embryos. Development 127, 1789–97. 18. Heasman J. (2002) Morpholino oligos: mak- ing sense of antisense? Dev. Biol. 243, 209–14. 19. Moody S.A. (2000) Cell lineage analysis in Xenopus embryos. Methods Mol. Biol. 135, 331–47. 20. Hartley R.S., Sible J.C., Lewellyn A.L., and Maller J.L. (1997) A role for cyclin E/Cdk2 in the timing of the midblastula transition in Xenopus embryos. Dev. Biol. 188, 312–21. 21. Hartley R.S., Rempel R.E., and Maller J.L. (1996) In vivo regulation of the early embryonic cell cycles in Xenopus. Dev. Biol. 173, 408–19. 22. Kim S., Li C., and Maller J. (1999) A mater- nal form of the phosphatase Cdc25A regu- lates early embryonic cell cycles in Xenopus laevis. Dev. Biol. 212, 381–91. 23. Murakami M.S., and Woude G.F.V. (1998) Analysis of the early embryonic cell cycles of Xenopus; regulation of cell cycle length by Xe-wee1 and Mos. Development 125, 237–48. 24. Kappas N.C., Savage P., Chen K.C., Walls A.T., Sible J.C. (2000) Dissection of the XChk1 signaling pathway in Xenopus laevis embryos. Mol. Biol. Cell 11(9), 3101–8. Expression of Exogenous mRNA in Xenopus 15
- 35. Chapter 2 Use of Luciferase Chimaera to Monitor PLCz Expression in Mouse Eggs Karl Swann, Karen Campbell, Yuansong Yu, Christopher Saunders and F. Anthony Lai Abstract The microinjection of cRNA encoding phospholipase C (PLC zeta) causes Ca2+ oscillations and the activation of development in mouse eggs. The PLC protein that is expressed in eggs after injection of cRNA is effective in causing Ca2+ oscillations at very low concentrations. In order to measure the amount and timecourse of protein expression we have tagged PLC with firefly luciferase. The expression of the luciferase protein tag in eggs is then measured by incubation in luciferin combined with luminescence imaging, or by the lysis of eggs in the presence of Mg-ATP and luciferin in a luminometer. The use of luciferase to monitor protein expression after injection of cRNA is a sensitive and effective method that efficiently allows for sets of eggs to be used for PLC quantitation, Ca2+ imaging, and studies of embryo development. Key words: Luminescence, luciferase, phospholipase, egg. 1. Introduction Mammalian eggs are large cells (100 mm in diameter) and readily amenable to microinjection. We have used pressure-based micro- injection as a means of introducing molecules into mouse eggs for many years. Our particular interest over the last few years has been focused on the role of a sperm-specific phospholipase C (PLC zeta) in causing the Ca2+ changes that lead to egg activation in mammals. This protein can trigger repetitive Ca2+ oscillations that are very similar to those seen at fertilization in mouse, pig, and human eggs (1). We have proposed that PLC is the ‘‘sperm factor’’ that is delivered by the sperm into the egg following David J. Carroll (ed.), Microinjection: Methods and Applications, Vol. 518 Ó 2009 Humana Press, a part of Springer ScienceþBusiness Media, LLC DOI 10.1007/978-1-59745-202-1_2 17
- 36. gamete fusion (2). It has been shown that recombinant PLC can cause Ca2+ release when it is injected into mouse eggs (3). We, and others, have also carried out biochemical studies of recombi- nant PLC (3,4). However, one of the major problems of working with recombinant PLC protein is that its Ca2+ oscillation-indu- cing activity is very labile, consistent with our observation that the PIP2 hydrolysis enzymatic activity is difficult to maintain. Conse- quently, we have used cRNA injection as a general means of introducing PLC into mouse eggs. The injection of cRNA for PLC also has the advantage that no protein contaminants are introduced into cells, in contrast to native protein isolation pro- cedures. One of the disadvantages of injecting cRNA PLC is that the level of expressed protein cannot be readily determined in living cells. An effective way to measure how much protein is being expressed is to inject cRNA for PLC that has been tagged with firefly luciferase (4). Using luciferase luminescence to measure protein expression is a highly sensitive technique. Luminescence has an advantage over the use of fluorescence-based methods in that it does not suffer from interference from auto-fluorescence which is quite considerable in mammalian eggs (5). The issue of sensitivity is particularly important since mouse PLC is active in mouse eggs at concentrations of 1–10 nM (3), and this very low level of protein is on the limit of detection for the most sensitive fluorescent proteins used in eggs (6). Furthermore, since human and monkey PLC appear to be more potent in causing Ca2+ oscillations in eggs than the mouse PLC (7), the physiological levels of PLC expression in many species may be undetectable using fluores- cently tagged PLC. The chief disadvantage in using luciferase luminescence is that the localization of the expressed protein is poor compared to fluorescence probes, where high-resolution confocal imaging can be used. However, with photon imaging cameras it is certainly possible to identify which individual eggs, or cells, are expressing luciferase and it is possible to quantitatively estimate how much luciferase-tagged protein is being expressed. In this chapter, we describe how we study the effects of PLC, and other PLC isoforms, or various mutant versions of PLCs, by injection of cRNA encoding luciferase-tagged versions of the proteins. In many experiments, we inject the cRNA and then measure Ca2+ signals from eggs for several hours after injection, before calibrating luciferase expression at a set time-point. In other cases, we monitor Ca2+ oscillations and luciferase expression from eggs and then place them in culture for further studies on their development. The methods we describe for PLC can be readily applied to other proteins and, for example, we have also injected and studied cyclin B levels in mouse eggs using a lucifer- ase-tagged cRNA (unpublished data). 18 Swann et al.
- 37. 2. Materials 2.1. DNA and RNA Preparation 1. To produce sufficient quantities of DNA plasmid, we use Qia- gen’s Plasmid Maxi Kit. Restriction enzymes are routinely purchased from New England Biolabs. To produce polyade- nylated cRNA, mMessage mMachine T7/T3/SP6 kits and Poly (A) Tailing Kits are used, along with SUPERase-In RNAse Inhibitor (Ambion). Rabbit Reticulocyte Lysate (Pro- mega) is used to assess RNA quality. 2. RNAse-free solutions and plasticware are prepared using treat- ment with diethyl pyrocarbonate (DEPC, Sigma). 3. Media for mouse eggs consists of M2 and acidified Tyrode’s solution (Sigma). We also use KSOM and a Hepes version of KSOM (HKSOM) which is made from stock using embryo- tested chemicals (Sigma) and clinical-grade water. The constitu- ents of KSOM and HKSOM are given in reference (8) and (9). Hyaluronidase M2 and acid Tyrodes solution are stored in ali- quots at 20°C. The M2 or HKSOM media are stored at 4°C and used for 2–3 weeks. For all imaging studies, firefly luciferin is added to HKSOM media. The luciferin (L6882 from Sigma) is made up at 100 mM in distilled water and stored in the 20°C freezer for 1 month. It is diluted into HKSOM shortly before use to give a final concentration of 100 mM (see Note 1). 4. The injection buffer consists of KCl/Hepes (120-mM KCl, 20-mM HEPES, pH 7.2). The buffer is made up in plastic vessels and then mixed with 1% Chelex 100 beads (Sigma) for 1 h (to remove divalent cations) before being filter-sterilized. For experiments where intracellular Ca2+ is to be measured Oregon Green BAPTA dextran (OGBD) (Molecular Probes, www.probes.com) is added to the injection buffer. Aliquots of injection buffers are stored in the 20°C freezer. 2.2. Mouse Eggs 1. We regularly use the MF1 strain of mice, but have obtained similar results with other strains of mice such as CD-1, or with F1 hybrid cross strains. The hormones were purchased from Dunlops Veterinary Supplies (www.dunlops.com). The super- ovulation of mice and collection of eggs is described in labora- tory manuals (10). 2. Eggs were manipulated in M2 media (Sigma) using fine-bore glass pipettes that were pulled in a flame to a diameter of approximately 80–100 mm. 2.3. Microinjection 1. For microinjection, borosilicate glass capillaries (GC150F, Harvard Apparatus Ltd., 1.5-mm outer diameter and 0.86- mm inner diameter) with an internal filament were pulled on a Luciferase Tag Expression in Mouse Eggs 19
- 38. vertical pipette puller (Model P-30; Sutter Instruments). The pipettes used for injection should be checked for appropriate tip size. This can be done by finding the minimal pressure required to blow bubbles in ethanol (11). Injection needles are backfilled with sterile microloader pipette tips (eppendorf). 2. Injection needles are clamped in a holder with a silver wire and side port (World Precision Instruments Inc, www.wpi-eur- ope.com). The holder is plugged into a preamplifier that is electrically connected to an intracellular amplifier (e.g., Electro 705 or Cyto 721; WPI). The preamplifier is held in the micromanipulator. 3. Pressure is applied to the back of the needle by pulses from a pressure injection system (Picopump, WPI) connected to the side port of the needle holder with stiff silicone tubing. 4. Mouse eggs are injected while being held by a ‘‘holding’’ pipette (Hunter Scientific) using suction via a syringe system (Narashige) containing embryo-tested mineral oil (Sigma). 5. The preamplifier and needle holder, and the ‘holding pipette’, are mounted on hydraulic manipulators (Narashige) that are fixed to the inverted microscope (TE2000, Nikon UK Ltd). 2.4. Imaging and Quantifying Luciferase 1. For imaging, the eggs are maintained in drops of media in a heated chamber (Intracel Ltd.) on the stage of an inverted fluorescence microscope (either a Nikon TE2000, or Zeiss Axiovert S100). Each microscope has the facility to direct 100% of the light from the eggs to the camera via either a side port or the base port. 2. The light collected from injected eggs is imaged using photon- counting imaging cameras. The cameras we currently use are cooled intensified CCD cameras (Photek Ltd.; www.photek.- com). Photek’s software is used for data collection and analysis (see Note 1). 3. The imaging systems are contained in a lightproof dark box. In one case, we have the facility to direct bright field or fluorescent illumination to the eggs via fiber optical cables. The gating of these light sources is controlled via Photek’s software that controls electro-mechanical shutters (Uniblitz; www.uniblitz.com) that are integrated into the light box. In another case, the microscope light sources are inside the box and simply switched off during luminescence imaging (see Note 2). 4. After imaging, the eggs can be lysed in order to quantify luciferase expression. This is done in a lysis buffer using a custom-made luminometer (see reference (12)). This essen- tially consists of a dark-proof tube holder that is adjacent to a cooled photomultiplier tube (S20 type tube, with a 20 Swann et al.
- 39. cooled housing and amplifiers from Electron Tubes Ltd.; www.electrontubes.com). The light output is measured using photon-counting discriminators and amplifiers with software supplied by Electron Tubes Ltd. Commercially available tube luminometers would also be suitable. 5. The lysis buffer consists of phosphate-buffered saline with 1- mM MgATP and 100-mM luciferin. Eggs are lysed with Triton X-100 and the amount of luciferase calibrated with recombi- nant luciferase protein. All these reagents are purchased from Sigma. 3. Methods 3.1. Synthesis of cRNA 3.1.1. Preparation of DNA 1. RNAse-free solutions and plasticware are prepared by incuba- tion in a solution of 0.1% DEPC overnight in a fume hood. Following this incubation, residual DEPC is removed by auto- claving. We routinely generate microgram quantities of the required DNA plasmid using Qiagen’s Plasmid Maxi Kit, fol- lowing the manufacturer’s instructions (a standard molecular biology/microbiology textbook (13) provides further infor- mation on Escherichia coli strains, transformation, and hand- ling). Depending on the plasmid copy number, 500 ml of DNA with a concentration of 0.5–2 mg/ml can be harvested from 250 ml of culture. 2. Due to the high processivity of RNA polymerases, it is necessary to linearize the circular plasmid DNA to prevent the production of extremely long RNA molecules. This can be achieved by digestion with a suitable restriction enzyme, obviously avoiding those which cut between the promoter and gene of interest. A 100-ml restriction digest containing 10 mg of DNA and 20 U of enzyme is incubated at a suitable temperature overnight. Complete linearization can be confirmed by running 2 ml on an agarose gel if necessary. 3. The linearized DNA is cleaned up by phenol/chloroform extraction. Essentially, an equal volume of TRIS-buffered phe- nol:choloroform:isoamylalcohol (25:24:1 v/v/v) is added and mixed by vigorous inversion for 30–60 s. The phases are sepa- rated by microcentrifugation at 14000 g for 3 min and the top, aqueous phase is transferred to a DEPC-treated microfuge tube using a DEPC-treated tip, taking care not to transfer any of the proteinaceous, white interphase. This extraction is repeated twice more. Luciferase Tag Expression in Mouse Eggs 21
- 40. 4. The DNA is precipitated by addition of 80 ml of isopropanol and 18 ml of 3-M sodium acetate, pH 5.2 and, following an incubation at 80°C for 1 h, is pelleted by microcentrifugation at 14000 g for 20 min at 4°C. The supernatant is removed and the pellet washed with 80% ethanol. Following a further 20- min spin, the pellet is left to air-dry for 10–15 min. 3.1.2. Translation and Polyadenylation of RNA 1. The DNA pellet is resuspended in 6 ml of nuclease-free water containing 20 U of SUPERase-In RNAse Inhibitor. This is then transferred to a fresh microcentrifuge tube, and a tran- scription reaction assembled at room temperature by adding 10 ml of ice-cold 2xNTP/CAP mix, 2 ml of room-temperature 10X reaction buffer, along with 2 ml of enzyme. 2. The reaction is then incubated at 37°C for 2 h. Addition of a poly-A tail, to enhance RNA longevity, is achieved by addition of 36-ml nuclease-free water, 20-ml E-PAP buffer, 10-ml 25- mM MgCl2, 10-ml 10-mM ATP, and 4-ml E-PAP to the 20 ml transcription reaction. The reaction is again incubated at 37°C for a further 2 h. 3. The polyadenylated RNA is precipitated by addition of 150-ml lithium chloride precipitation solution, and incubated at 80°C for 1 h. The RNA is pelleted by 4°C microcentrifuga- tion at 14000 g for 30 min. The supernatant is discarded, the pellet washed with 80% ethanol, and further centrifuged for 15 min. Following air-drying, the RNA is resuspended by addition of 9 ml of nuclease-free water and 1 ml (20 U) of SUPERase-In RNAse Inhibitor. 3.1.3. Quantification and Dilution of RNA 1. The RNA is quantified by measuring its absorbance at 260 nm in a 500-ml quartz cuvette. We routinely use 1 ml of sample, giving a dilution factor of 1 in 500. The amount of RNA is then calculated using the standard equation: RNA conc ðmg=mlÞ ¼ 40 A260 500 2. The RNA can stored at 80°C in 1-ml aliquots. We commonly store RNA as either undiluted stock aliquots, or working ali- quots diluted to 2, 0.2, or 0.02 mg/ml. When diluting, we commonly add 20U SUPERase-In to the RNA prior to aliquotting. 3. Just before injection, the RNA is mixed with other compo- nents, such as OGBD, and then kept on ice for the period over which injections are carried out (1 h). If there is any remain- ing solution in the aliquots after injection, it is discarded and a fresh aliquot is used for each injection session. 22 Swann et al.
- 41. 3.1.4. Assessment of RNA Quality Initially, it may be necessary to check batches of RNA for signs of RNAse contamination, which leads to degradation of the RNA. Commonly this can be achieved by running an aliquot of RNA on a denaturing agarose gel, checking for the presence of a single species of defined size without a smear of lower-molecular-weight frag- ments, indicative of RNA degradation. However, due to the het- erogeneity in size of the poly-A tail, a single species is rarely seen, leading to some degree of uncertainty about the quality of the RNA. Instead, we check that the RNA can be translated into a protein of the predicted molecular weight. This is achieved using 1–2 mg of RNA in a Rabbit Reticulocyte Lysate reaction (Promega). We label the protein with [35 S]-Pro-Mix, and, following separation on SDS-PAGE, use autoradiography to determine its molecular weight. Alternatively, we have also satisfactorily used ‘‘cold’’ methionine in the reaction, and then used antibodies to detect the protein of interest on a western blot following the SDS-PAGE. 3.2. Microinjection of Eggs 1. Zona intact mouse eggs are placed in a shallow drop (1 ml) of media covered in oil, in the lid of a petri dish. The dish is placed on the microscope stage without heating. A silver wire is placed in the injection drop and this wire is held in place via a small manipulator. This wire is connected to a longer standard cop- per wire to the chassis ground of the electrical amplifier. This wire allows for an electrical circuit to form between the ground and the tip of the pipette once it is place in the media (a circuit is indicated by the amplifier). If this does not occur, then the pipette should be replaced because the tip is probably blocked. 2. The RNA solution to be injected should be spun (13,000 rpm in a benchtop microcentrifuge) for several minutes before injection. For RNA injection, we generally use tips of 0.75–0.9 mm in diameter. The injection pipettes are backfilled with 1 ml of injection solution containing the RNA. 3. This pipette is then held in the specialized holder (containing a silver wire) that is then fitted onto the preamplifier that is itself clamped into the micromanipulator. 4. For experiments where fluorescence is also measured, to look at changes in Ca2+ dynamics within the egg, the luciferase- tagged cRNA is mixed with an equal volume of 1-mM OGBD prepared in KCl Hepes pH 7.2. Even if a Ca2+ dye is not to be used, it is useful to mix the RNA solution 1:1 with KCL Hepes buffer so as to have some salts present in the injection buffer. 5. For injection, each egg is held by suction with the holding pipette and then the tip of the injection pipette is manipulated so that it will touch the plasma membrane. The injection pipette is then pushed into egg in a way that deforms the zona pellucida. At some point, the zona will jump back into Luciferase Tag Expression in Mouse Eggs 23
- 42. shape, which is a sign that the zona pellucida is penetrated. At this point, the negative capacitance is applied to the amplifier that is connected to the back of the specialized pipette holder. This causes the pipette tip to enter the cytoplasm. The operator should then make sure that the tip of the injection pipette is in focus and a pressure pulse is applied from the picopump to push a bolus of solution into the egg. 6. The pressure pulses we use are typically 100 ms1 s long, at a pressure of 20 psi. The volume of solution injected is esti- mated by the diameter of cytoplasmic displacement caused by the injection and should correspond to 3–5% of the egg volume. In practice, the first egg can be used for a test with the pulse duration and pressure being adjusted to suit the amount of solution injected. Once familiar with this system, it is possible to inject 30 eggs in 20 min. However, tips often become blocked during injection and need to be replaced. 3.3. Imaging of Luciferase Luminescence 1. After injection, the eggs are placed in drops of media for imaging. In most experiments, the eggs are left zona intact and placed in a small (50 ml) drop of HKSOM media, which is under mineral oil in a heated (37°C) chamber with a glass coverslip that sits on the inverted microscope. The HKSOM media contains BSA (4 mg/ml) and 100-mM luciferin (see Note 3). Figure 2.1 shows the luminescence from a single mouse egg injected with cRNA for luciferase alone. The time- course of luciferase expression lasts at least 10 h with a peak at around 4–5 h post-injection. 2. For some experiments where we want to add extra compounds, or sperm, during the course of imaging, the eggs have to be stuck down. We do this by briefly treating the eggs with acid Tyrode’s solution to remove the zona pellucidas and then immediately placing the eggs in 1-ml drop of the HKSOM in a chamber that has a polylysine-coated (1 mg/ml) coverslip. 0 10000 5 Hours Photons per minute Fig. 2.1. Luminescence (in photon counts per minute) from a mouse egg injected with luciferase cRNA. The egg was injected with 10 pl of 2 mg/ml of cRNA and imaged in media in the presence of 100-mM luciferin 10–20 min after injection. 24 Swann et al.
- 43. 3. To image intracellular Ca2+ in eggs (injected with OGBD), we monitor fluorescence for the period during which Ca2+ signals occur (5 h). At the end of this period, the luminescence is then measured on the same set of eggs (see Note 4). 4. Fluorescence or luminescence is imaged in the eggs using either 20x 0.65 NA or 10x 0.5 NA Fluor objectives. The light (100%) is directed via either a sideport or baseport to the ICCD camera. We use the same ICCD camera to monitor both fluorescence and luminescence. The main difference is that during fluorescence measurements, the eggs are exposed to excitation light. This means that a standard fluorescence filter block is in place to enable epifluorescence illumination. For OGBD we use a FITC block or else a modified block with a 500-nm longpass filter. Figure 2.2a shows the relative changes in fluorescence from eggs injected with OGBD and PLC-luc cRNA. The spike-like increases indicate intracellular Ca2+ oscillations, as described previously. 1 2 30 min Fluorescence (a.u.) Luminescence (photons per minute) egg 1 egg 2 5 Hours 250 0 a) b) c) Fig. 2.2. Mouse eggs injected with PLC-luc cRNA. Eggs were injected with 10 pl of 0.2 mg/ml cRNA. In (a) the fluorescence (in arbitrary units, a.u.) of Oregon Green BAPTA dextran is shown. The oscillations in fluorescence indicate intracellular Ca2+ oscillations are occurring. In (b) the luminescence from two other eggs injected with PLC-luc cRNA and incubated in 100-mM luciferin is shown. The luminescence is recorded continuously with this experiment. In (c) an image of the group of eggs injected with PLC-luc cRNA is shown for different time periods. The arrows point to eggs 1 and 2 that are shown in (b). Luciferase Tag Expression in Mouse Eggs 25
- 44. 5. The excitation light source is from a halogen lamp (see Note 5). Fluorescence is monitored in injected eggs for 4 h or 5 h, and then by measuring the OGBD fluorescence with low-level excitation light, the luminescence is measured from the same set of eggs by recording the light from eggs with the excitation light turned off. During the fluorescence recording, the lumi- nescence signal may increase slightly and so the recorded fluor- escence signal actually contains a small component of luminescence. However, the fluorescence signals are typically more than 100 times greater than the luminescence signals, so can be ignored in practice. During fluorescence recording, the camera’s sensitivity can be reduced to 10%. 6. At the end of the fluorescence measurements, the same set of eggs are then monitored for luminescence by integrating light emission (in the absence of fluorescence excitation) for 20 min using the same ICCD camera. The cameras we use, typically have a very low background count such that the background noise from an area the size of one egg is about 1 photon per minute. The luminescence signal starts to increase above back- ground within 10 min of the start of recording (Fig. 2.2). The signal then continues to increase for several hours and does not start to decrease until about 8–10 h. The level of signal depends upon the amount of cRNA injected and the particular construct used (see Note 6). Figure 2B and C shows the images and luminescence integrated from different time periods, as well as the timecourse of expression from two of the eggs in the group that illustrate the range of variation in luminescence. 7. If the experiment only requires a measure of the relative amounts of lufciferase expression, then zona intact eggs can be removed from the imaging drop and placed in KSOM media in drops under oil in a 37°C 5% CO2 incubator. If an absolute calibration of expression is required then the eggs are lysed in a luminometer. 3.4. Quantifying Luciferase Expression in a Luminometer 1. Imaging the luciferase luminescence from eggs can ensure that all eggs counted in an experimental group express the lucifer- ase-tagged PLC. To measure the amount of luciferase protein expressed, groups of eggs are collected from the imaging drop and then lysed in a buffer in a luminometer (see Note 7). For each experiment, groups of eggs, verified as being luminous, are collected and placed in a test tube containing phosphate- buffered saline with 1-mM Mg2+ ATP and 100-mM luciferin. 2. The eggs are then lysed with 0.5% Triton X-100 and the steady- state light compared to that emitted from serial dilutions of recombinant firefly luciferase (Sigma) in the same buffer. The amount of luciferase activity measured for each group of eggs is then divided by the number of luminous eggs to obtain the 26 Swann et al.
- 45. mean value for protein expression of each type of PLC-lucifer- ase. We have found that injection of mouse PLC-luciferase into mouse eggs can lead to Ca2+ oscillations and the expression of 0.1–0.2 pg of protein in a 4-h period following injection (4). 4. Notes 1. We have previously used an imaging photon detector (IPD) system which was set up by, and used software from, Science- wares (www.sciencewares.com). The IPD camera (Photek Ltd.) uses a different principal for light collection from the ICCD cameras. We have not noticed any significant difference in sensitivity of these two types of photon-counting detectors. 2. It is essential that some form of dark box is constructed around the microscope. The light level in a typical darkened room, where standard fluorescence microscopy is carried out, is usually much too high and causes considerable interference when ima- ging luminescence. Any light sources within the darkbox should be removed or covered with black tape. If the microscope used is motorized in any way, it will probably be necessary to switch it off completely during luminescence imaging since internal LEDs will cause an elevated background light. 3. We use 100 mM for mouse eggs, but a range of luciferin con- centrations (1 mM to 1 mM) are cited for use in luciferase- imaging of cells in general. The higher concentrations are not always the most effective, because the luciferin luciferase reac- tion shows ‘‘flash kinetics’’ which means that the reaction rate can decrease with time due to inhibition from the reaction product, oxyluciferin (12). The best concentration to use can depend upon a range of factors that are specific to a cell type, and it is best to test different concentrations. The luciferase also depends upon ATP and can be used to monitor ATP concen- trations in eggs (5, 14). For monitoring the timecourse of expression this is not a major issue because the changes in luminescence caused by ATP increases in activating eggs accounts for a 10–20% change in the total light output, and this is hardly detectable in studies using effective amounts of PLC-luc, where the luminescence signals are about 1–10 photons per second for each egg. In order to effectively measure the ATP change at fertilization, we have to inject high concentrations of recombinant luciferase protein, which results in luminescence values of 100–1000 photons per second per egg (14). 4. We use OGBD to measure Ca2+ because the imaging systems we use can only monitor fluorescence at a single wavelength. Luciferase Tag Expression in Mouse Eggs 27
- 46. Consequently, a dye such as OGBD is used, since it undergoes an increase in fluorescence intensity with an increase in Ca2+ , and being dextran-linked it does not undergo compartmenta- lization which can be a problem in mouse eggs (15). Other dyes, such as fura 2, which permits ratio excitation, could be used in conjunction with luciferase monitoring. However, it is worth noting that luciferin is fluorescent when excited with UV light, so if fura 2 is to be used, the luciferin should not be added to the media until the fluorescence imaging is finished. 5. A halogen light source with a stabilized power supply is used because this can easily be reduced to the minimum level required. The excitation light used with photon-counting cam- eras is generally much lower than with standard cooled CCD cameras and so the use of Xenon lamp (for example) with multi- ple neutral density filters creates unnecessary light and heat. 6. It is important to optimize the length of the spacer residues present between the protein of interest and luciferase, as we have found that this can alter the expression level of the pro- tein. This may be related to the potential for changes in the protein secondary structure, consequent to tagging with luci- ferase, and will vary with each protein. For PLCs, we find that a spacer of four residues works well. 7. The relative expression of a PLC can be easily assessed and this can be used for studies on egg activation, or for studies on the effects of PLC on later embryo development. However, it is not simple to calibrate the absolute amount of protein expressed in single eggs using the luminescence from living eggs on the microscope. This is partly because the light emitted from firefly luciferase depends upon ATP and pH. Consequently, the pre- cise level of free ATP and pH in an egg would have to be known to calibrate the absolute amount of luciferase. Acknowledgments Our work is supported by the BBSRC, Wellcome Trust, and Cardiff Partnership Fund. References 1. Swann, K., Saunders, C.M., Rogers, N. and Lai, F.A. (2006) PLC (zeta): A sperm pro- tein that triggers Ca2+ oscillations and egg activation in mammals. Sem. Cell Dev. Biol. 17, 264–73. 2. Saunders, C.M., Larman, M.G., Parrington, J., Cox, L.J., Royse, J., Blayney, L.M., Swann, K. and Lai F.A. (2002) PLC: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development. Development 129, 3533–44. 28 Swann et al.
- 47. 3. Kouchi, Z., Fukami, K., Shikano, T., Oda, S., Nakamura, Y., Takenawa, T and Miya- zaki, S. (2004) Recombinant phospholipase Czeta has high Ca2þ sensitivity and induces Ca2þ oscillations in mouse eggs. J. Biol. Chem. 279, 10408–12. 4. Nomikos, M., Blayney, L.M., Larman, M.G., Campbell, K., Rossbach, A., Saunders, C.M., Swann, K. and Lai, F.A. (2005) Role of phos- pholipase C-(zeta) domains in Ca2+ -depen- dent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca2+ oscillations. J. Biol. Chem. 280, 31011–18. 5. Dumollard, R., Marangos, P., Fitzharris, G., Swann, K., Duchen, M. and Carroll, J. (2004) Sperm-triggered [Ca2+ ] oscillations and Ca2+ homeostasis in the mouse egg have an absolute requirement for mitochon- drial ATP production. Development 131, 3057–67. 6. Yoda, A, Oda, S., Shikano, T., Kouchi, Z., Awaji, T., Shirakawa, H., Kinoshita, K and Miyazaki, S. (2004) Ca2+ oscillation-indu- cing phospholipase C zeta expressed in mouse eggs is accumulated in the pronucleus during egg activation. Devel. Biol. 268, 245–57 7. Cox, L.J., Larman, M.G., Saunders, C.M., Hashimoto, K., Swann, K. and Lai, F.A. (2002) Sperm phospholipase C from humans and cynomolgus monkeys triggers Ca2+ oscillations, activation and develop- ment of mouse oocytes. Reproduction 124, 611–23. 8. Summers, M.C., Bhatnagar. P.R., Lawitts. J.A., Biggers. J.D. (1995) Fertilization in vitro of mouse ova from inbred and outbred strains: complete preimplantation embryo development in glucose-supplemented KSOM. Biol. Reprod. 53, 431–7. 9. Summers, M.C., McGinnis, L.K., Lawitts, J.A., Raffin, M., Biggers, J.D. (2000) IVF of mouse ova in a simplex optimized medium supplemented with amino acids. Hum. Reprod. 15, 1791–801. 10. Hogan, B., Costantini, F. and Lacy, E. (1986). Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbour Laboratory, Cold Spring Harbor, NY. 11. Schnorf, M., Potrykus, I. and Neuhaus, G. (1994) Microinjection technique: routine system for characterization of microcapil- laries by bubble pressure measurement. Exp. Cell. Res. 210, 260–7. 12. Campbell, A.K. (1988) ‘Chemiluminescence’ Ellis Horwood Series on Biomedicine. 13. Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 14. Campbell, K. and Swann, K. (2006) Ca2+ oscillations stimulate an ATP increase during fertilization. Devel. Biol. 298, 225–53. 15. Carroll, J., Swann, K., Whittingham, D. and Whitaker, M.J. (1994) Spatiotemporal dynamics of intracellular [Ca2+ ]i oscillations during growth and meiotic maturation of mouse oocytes. Development 120, 3507–17. Luciferase Tag Expression in Mouse Eggs 29
- 48. Chapter 3 Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino Oligos Jeffrey M. C. Lau and Anthony J. Muslin Abstract The 14-3-3 intracellular phosphoserine/threonine-binding proteins are adapter molecules that regulate signal transduction, cell cycle, nutrient sensing, apoptotic, and cytoskeletal pathways. There are seven 14- 3-3 family members, encoded by separate genes, in vertebrate organisms. To evaluate the role of individual 14-3-3 proteins in vertebrate embryonic development, we utilized an antisense morpholino oligo micro- injection technique in Xenopus laevis embryos. By use of this method, we showed that embryos lacking specific 14-3-3 proteins displayed unique phenotypic abnormalities. Specifically, embryos lacking 14-3-3 t exhibited gastrulation and axial patterning defects, but embryos lacking 14-3-3 g exhibited eye defects without other abnormalities, and embryos lacking 14-3-3 appeared completely normal. These and other results demonstrate the power and specificity of the morpholino antisense oligo microinjection technique. Key words: Morpholino, Xenopus, microinjection, embryogenesis, 14-3-3. 1. Introduction 14-3-3 proteins are intracellular dimeric phosphoserine-binding molecules that regulate important aspects of cell physiology (1–3). 14-3-3 family members participate in signal transduction, cell cycle, apoptotic, metabolic, and cytoskeletal pathways. In vertebrate organisms, there are seven 14-3-3 family members encoded by separate genes, named 14-3-3 , g, , , , t, and (2). When tested in vitro, all seven family members bind with similar affinity to phosphoserine-containing peptides containing the RSxSxP motif, where x is any amino acid (4,5). Despite their similar binding properties in vitro, each family member has a unique expression pattern in embryonic development, and there David J. Carroll (ed.), Microinjection: Methods and Applications, Vol. 518 Ó 2009 Humana Press, a part of Springer ScienceþBusiness Media, LLC DOI 10.1007/978-1-59745-202-1_3 31
- 49. may be a secondary contact point that differentiates the binding properties of individual 14-3-3 proteins in vivo (6,7). In previous work, we investigated the role of 14-3-3 proteins in Xenopus laevis development by use of a peptide inhibitor of all 14-3-3 proteins, the R18 peptide (8). We injected RNA encoding the R18 peptide linked to glutathione S-transferase (GST). Microinjection of GST-R18 into two-cell embryos resulted in major phenotypic abnormalities, including reduced mesoderm induction with abnormal gastrulation. These and other experi- ments showed that global 14-3-3 inhibition blocked FGF- mediated mesodermal differentiation and patterning. To test the role of individual 14-3-3 proteins in early Xenopus development, we used a morpholino antisense oligo microinjection technique (6). In this method, morpholinos that target individual Xenopus 14-3-3 family members were injected into two-cell-stage embryos. The ability of the morpholinos to specifically knockdown target protein levels was documented by analysis of embryonic protein lysates by Western blotting with family member-specific anti-14-3-3 antibodies. Phenotypic abnormalities in injected embryos were evaluated by visual inspection and by analysis of gene expression by whole-mount in situ hybridization. These experiments demonstrated that embryos that were lacking 14-3-3 t, and to a lesser degree 14-3-3 , exhibited gastrulation and axial patterning defects, but embryos lacking 14-3-3 exhibited eye defects without other abnormalities. Embryos lacking 14-3-3 appeared completely normal. Therefore, individual 14-3-3 proteins have distinct roles in the regulation of Xenopus development. 2. Materials 2.1. Sexually Mature Xenopus laevis 1. African clawed frogs, Xenopus laevis, are obtained from Xeno- pus One (Dexter, MI, http://www.xenopusone.com), Xenopus Express (Brooksville, FL, http://www.xenopus.com), or Nasco (Modesto, CA, http://www.enasco.com). 2. Wild-type sexually mature males (7.5–9 cm) and females (9–14 cm) are used for in vitro fertilization experiments. For whole-mount in situ hybridization and immunostaining experiments, sexually mature albino males and females are used to obtain non-pigmented embryos. 2.2. Morpholino Antisense Oligos 1. Morpholinos were purchased from GeneTools, LLC (Philo- math, OR). Morpholinos are designed to be antisense to the initiation AUG and subsequent 22 ribonucleotide residues in the mRNAs encoding specific Xenopus proteins (see Note 1). 32 C. Lau and Muslin
- 50. 2. Sequence of the specific morpholinos are the following: 14-3-3 morpholino, 5-TCTGTACCAGTTCACTCTTGTCCAT-30 ; 14-3-3 morpholino, 50 -GCAACTGCTGCTCCCGATCA- GCCAT-30 ; 14-3-3 morpholino, 50 -ACACTAAATCCTCT CGCTCTTCCAT-30 , and 50 -TACACTAAATCCTCTCGCTC TTCCA-30 ; 14-3-3 g morpholino, 50 -GCACCAGCTGCTCG CGGTCCACCAT-30 ; 14-3-3 t morpholino, 50 -TCTGGATTT GTGCGGTCCTGTCCAT-30 , and 50 -GTCTGGATTTGTGC GGTCCTGTCCA-30 ; and 14-3-3 morpholino, 50 - TCTGGA CCAGTTCATTTTTATCCAT-30 . The control morpholino is a scrambled version of the experimental morpholino commercially available from GeneTools, LLC (Philomath, OR). 3. Morpholinos are re-constituted in RNAase-free distilled water to a final concentration of 1 mmol/ml and stored in aliquots at 80°C. 2.3. Family-Member- Specific Anti-14-3-3 Antibodies and Western Blotting 1. 14-3-3 family member-specific antibodies (Santa Cruz Bio- technology, Inc., Santa Cruz, CA) are used to detect specific knockdown of 14-3-3 family member protein levels on immunoblots. Total ERK protein level is measured with anti-p44/p42 antibody (#9102, Cell Signaling, Danvers, MA) as loading control. 2. Primary antibodies: 14-3-3 (A-15, sc#17288), (T-16, sc#1020), g (C-16, sc#731), (K-12, sc#17286), t (C17, sc#732), (C-16, sc#1019) primary antibody stock solutions are stored at 4°C. Working solutions are prepared by making a 1:500 dilution of the stock antibody in 1X TBS/T contain- ing 5% bovine serum albumin (#A9647, Sigma) and 0.05% sodium azide. Working solutions are stored at 4°C and can be re-used multiple times. 3. Primary antibody: p44/p42 total ERK antibody (#9102, Cell Signaling, Danvers, MA) is stored at 20°C. Working solu- tion is prepared by making a 1:1000 dilution of the stock antibody in 1X TBS/T containing 5% bovine serum albumin (#A9647, Sigma) and 0.05% sodium azide (Sigma). The working solution is stored at 4°C and can be re-used multiple times. 4. 10x TBS/T: 100-mM Tris-HCl pH 8.0, 1.5-M NaCl, 0.5% Tween 20. Dilute to 1X concentration with distilled water to make 1X working solution. Store both 10X and 1X solutions at room temperature. 5. Secondary antibody solution: Horseradish peroxidase (HRP)-conjugated secondary antibody derived from the correct species (Cell Signaling, Danvers, MA) is diluted 1:5000 in 10 ml of 1X TBS/T containing 5% fat-free dry milk powder. Analysis of 14-3-3 Family Member Function in Xenopus Embryos 33
- 51. 6. ECL Western blotting detection reagents (Amersham Bios- ciences, England) and ISO-MAX autoradiography/X-ray film (#GX-330810, Scimart, St. Louis, MO). 7. NP40 lysis buffer: 20-mM Tris HCl pH 7.5, 137-mM NaCl, 50-mM NaF, 0.5% NP40, 2-mM EDTA, 0.017 mg/ml apro- tinin, 1-mM phenylmethylsulfonyl fluoride (PMSF), 1-mM Na3VO4. Store at 4°C. Aprotinin (Sigma), PMSF, and Na3VO4 are added to the lysis buffer from 100x stock imme- diately before use. 8. PMSF 100x stock solution (100 mM): Dissolve 174.2 mg PMSF in 10-ml ethanol. Aliquot and store at 20°C. Add 1:100 to NP40 lysis buffer. 9. Na3VO4 100x stock solution (100 mM): Dissolves 184-mg Na3VO4 in 10-ml water, adjust to pH 10.0 with NaOH and HCl, boil until it turns colorless, and re-adjust pH to 10.0. Aliquot and store at 20°C. Add 1:100 to NP40 lysis buffer. 10. 2x SDS loading buffer: 950 ml Laemmli sample buffer (#161- 0737, BioRad, Hercules, CA) is combined with 50 ml beta- mercaptoethanol (#M3148, Sigma) to make 2x SDS loading buffer. Store at room temperature. 11. Western stripping buffer: We use either 0.2x NaOH solution or Restore Western blot stripping buffer (Pierce Biotechnol- ogy, Inc., Rockford, IL) and both are effective at completely removing signal from western membranes. 2.4. Removal of Testes 1. 20x Modified Barth’s Solution (MBS) Solution: 1.76-M NaCl, 20-mM KCl, 48-mM NaHCO3, 16.4-mM MgSO4, 200-mM Hepes, 6.6-mM Ca(NO3)24H2O, 8.2-mM CaCl26H2O, pH 7.4. Store the 20X stock solution at 4°C. Sterilize by filtration. Dilute in distilled water to make 1X or 0.1X working solutions. Working solutions are stored at room temperature. 2. Testes solution: 1X MBS containing 20% fetal calf serum (FCS, Sigma) and 1x penicillin/streptomycin. Sterilize by filtration. Store at 4°C. 3. Tricane methanesulfonate (MS-222, Sigma). 4. Penicillin/Streptomycin 100X stock solution (#P0781, Sigma). 2.5. Microinjector Apparatus 1. Microinjector (PLI-100, Harvard Medical Systems Corp, Greenvale, NY). 2. Joystick micromanipulator (MN-151, Narishige, Japan). 3. Micropipette puller (P30, Sutter Instrument Co., Greenvale, NY). 4. Filamented borosilicate glass capillaries (BF100-50-10, Sutter Instrument Co., Greenvale, NY). 34 C. Lau and Muslin
- 52. 5. Compact stereomicroscope (SMZ-2B, Nikon) with fiber optic cold light source (KL1500, Schott, Elmsford, NY). 2.6. In Vitro Fertilization of Embryos 1. Human chorionic gonadotropin (CG-10, Sigma) is dissolved in 10-ml sterile water to yield a final concentration of 1000 IU/ml and stored at 4°C. 2. Embryo medium: 0.2X MBS solution, 1X penicillin/strepto- mycin. Sterilize by filtration. Store at 4°C and warm up to room temperature before use. 3. Cysteine solution: Dissolve 2-g/L-cysteine (#168149, Sigma) in 100-ml distilled water, adjust to pH 7.6–7.8 with NaOH. Prepare immediately before use and keep at room temperature. 3. Methods 3.1. Removal of Xenopus Testes 1. Anesthetize a sexually mature male frog by placing it in a container with 0.3% tricane methanesulfonate (MS-222, Sigma) for 20 min. Euthanize by cervical translocation with a wire cutter. 2. Make a lower abdominal sagittal incision and remove the testes. The testes are cream-colored oval structures that are attached to the anterior, ventral surface of the kidneys. 3. Rinse the testes in 1X MBS and transfer to a 50-ml conical tube containing 10-ml testis solution. Store at 4°C. These testes can stay fresh up to 2 weeks for use in in vitro fertilization experiments. 3.2. Preparation of In Vitro Embryos 1. The evening prior to spawning, inject 800 IU of human chor- ionic gonadotropin (CG-10, Sigma) into the dorsal lymph sac of a sexually mature female frog. While eggs from one frog may be enough for an entire day’s experiments (up to 8000 eggs laid in a day), it may be best to spawn multiple frogs (we spawn three at a time) to ensure that at least one frog lays eggs of sufficient quality for in vitro fertilization experiments. 2. Female frogs are maintained between 14°C and 19°C over- night. It takes longer to start spawning if a frog is kept at a colder temperature. In general, it takes approximately 10–12 h to start spawning at 19°C, 11–13 h at 16°C, and 15 h at 14°C. 3. Once a frog starts spawning, collect the eggs into a 100-mm petri dish containing 1x MBS. 4. Cut off about one-quarter of a testis with a razor blade and homogenize it in 1-ml 1X MBS in a 1.5-ml eppendorf tube. Analysis of 14-3-3 Family Member Function in Xenopus Embryos 35
- 53. Other documents randomly have different content
- 54. a defect in the evidence; and I understand he always praises me up to the skies, and says I’m one of the best of men.’” The largest of all the classes of thieves, and that which employs the most extensive range of intellect, of age, and of dress, is the pickpocket. From the first-rate thief, who works about the banks for six or nine months until he gets a “good thing” to the miserable urchin who filches a pocket-handkerchief, how vast a descent! Although strung together by the common thread of crime, and pursuing, as it were, the same line of business, a duke could not, and certainly would not, look down upon a street-sweeper with half the hauteur that the leading rogues do upon the Fagin-led urchin who replenishes with bandanas the stalls of Field-lane. The popular notion of swellmobsmen is far wide of the truth. It is supposed that they may be at once recognized by a certain ultra-foppish manner of dressing, and an excess of jewellery, whereas the aim of a professor of the “conveying” art is to go about his occupation unobserved; for to be known to the police is to be disappointed of his booty. He has his clothes built by the most correct tailor, and gets himself up as much like a gentleman as possible. The necessities of his art, it is true, oblige him to carry a coat over his arm in all weathers; but so may any veritable man of fashion, without creating suspicion. Still, though he may manage to pass free in a crowd, and frequent fashionable assemblies without being suspected by the public, the professed thief-catcher is rarely to be deceived by appearances. As the hunter marks his quarry by peculiar signs known only to his craft, so the detective can at once ascertain whether the fine gentleman walking carelessly along is “wrong,” as the slang term is, or a respectable character. The principal sign by which a thief may be distinguished in any assembly is the wandering of his eye. Whilst those about him are either listening to a speaker or witnessing a spectacle, his orbits are peering restlessly, not to say anxiously around. When the thief-taker sees this, he knows his man. One of the detective police who attended at the laying of the foundation-stone of the Duke of
- 55. Wellington’s College, thus explained to us the capture of a gentlemanly-looking person who was present on that occasion:—
- 56. “If you ask me to give my reason why I thought this person a thief the moment I saw him, I could not tell you; I did not even know myself. There was something about him, as about all swellmobsmen, that immediately attracted my attention, and led me to bend my eye upon him. He did not appear to notice my watching him, but passed on into the thick of the crowd, but then he turned and looked towards the spot in which I was—this was enough for me, although I had never seen him before, and he had not, to my knowledge, attempted any pocket. I immediately made my way towards him, and, tapping him on the shoulder, asked him abruptly, ‘What do you do here?’ Without any hesitation, he said, in an under tone, ‘I should not have come if I had known I should have seen any of you.’ I then asked him if he was working with any companions, and he said, ‘No, upon my word, I am alone;’ upon this I took him off to the room which we had provided for the safe-keeping of the swellmobsmen.” This was a daring stroke, but it succeeded as it deserved. If the man had been really honest, he would have turned indignantly upon the person who questioned him; but pickpockets are essentially cowards, both morally and physically, and they generally come down at once to save trouble, when the officer has his eye upon them, as the opossums were wont to do when they espied that dead shot Colonel Crockett. There is a striking example of this weakness of their tribe in the amusing work of the “Englishwoman in America.” The scene is an American railway-carriage:— “I had found it necessary to study physiognomy since leaving England, and was horrified by the appearance of my next neighbour. His forehead was low, his deep-set and restless eyes significant of cunning, and I at once set him down as a swindler or pickpocket. My convictions of the truth of my inferences were so strong, that I removed my purse—in which, however, acting by advice, I never carried more than five dollars—from my pocket, leaving in it only my handkerchief and the checks for my baggage, knowing that I could not possibly keep awake the whole morning. In spite of my endeavours to the contrary, I soon sank into an oblivious state, from
- 57. which I awoke to the consciousness that my companion was withdrawing his hand from my pocket. My first impulse was to make an exclamation; my second, which I carried into execution, to ascertain my loss; which I found to be the very alarming one of my baggage-checks; my whole property being thereby placed at this vagabond’s disposal, for I knew perfectly well, that if I claimed my trunks without my checks, the acute baggage-master would have set me down as a bold swindler. The keen-eyed conductor was not in the car, and, had he been there, the necessity for habitual suspicion, incidental to his position, would so far have removed his original sentiments of generosity as to make him turn a deaf ear to my request, and there was not one of my fellow-travellers whose physiognomy would have warranted me in appealing to him. So, recollecting that my checks were marked Chicago, and seeing that the thief’s ticket bore the same name, I resolved to wait the chapter of accidents, or the reappearance of my friends.... With a whoop like an Indian war-whoop the cars ran into a shed—they stopped—the pickpocket got up—I got up too—the baggage-master came to the door: ‘This gentleman has the checks for my baggage,’ said I, pointing to the thief. Bewildered, he took them from his waistcoat- pocket, gave them to the baggage master, and went hastily away. I had no inclination to cry ‘Stop thief!’ and had barely time to congratulate myself on the fortunate impulse which had led me to say what I did, when my friends appeared from the next car. They were too highly amused with my recital to sympathize at all with my feelings of annoyance; and one of them, a gentleman filling a high situation in the East, laughed heartily, saying, in a thoroughly American tone, ‘The English ladies must be ‘cute customers’ if they can outwit Yankee pickpockets.’” The quickness and presence of mind of this lady was worthy of the practised skill of the detective who marked his man at the Wellington College ceremonial. That same gathering afforded another example of the cowardice of the swell mob. Immediately they came upon the ground, fourteen of them were netted before they had time to try the lightness of their fingers. They were confined in a single room
- 58. with only two policemen to guard them, yet they never attempted to escape, although their apprehension was illegal, but waited patiently until the crowd had dispersed. When the doors were thrown open, they immediately made a rush like so many rats from a trap, and never stopped until they were well out of sight of the police. The rapidity with which they bolted was caused by their desire to avoid being paraded before the assembled constables, a measure which is often taken by the police, in order that they may know their men on another occasion. If, however, the swellmobsman’s eye is for ever wandering in search of his prey, so also is that of the detective; and instances may occur when the one may be mistaken for the other. At the opening of the Crystal Palace, a party of detectives distributed among the crowd, observed several foreigners looking about them in a manner calculated to rouse their suspicions. These individuals were immediately taken into custody, notwithstanding their strong and vehement expostulations made in very good French. When brought before the inspector, it came out that they were Belgian police, sent over at the request of our Government to keep a look out on the mauvais sujets of their own nation. The swellmobsmen proper generally work together at races, in gangs of from three to seven; those who “cover,” as it is termed, making a rush to create pressure, in order that the pickpocket may use his hand without being noticed. In taking watches it is generally supposed that the ring is cut by a pair of wire-nippers. This is rarely the case; thieves have no time in operating to use any other implement than their own nimble fingers, and the ring of the watch is wrenched off with the utmost ease, as the purchase upon it is very great. A police magistrate, of large experience, suggests that the way to baffle the fraternity would be to make the ring work upon a swivel. Inferior classes of thieves work in smaller “schools,” say of a couple of women and a boy, whose little hand is capitally adapted for the work. Whilst one woman pushes, the lad attempts the pocket of the person nearest him, and the third “watches it off,” as it is called; if she observes that the youth’s attentions have been noticed, she immediately draws him back with a “Ha, Johnny, why do you
- 59. push the lady so!” Look to your pockets, good reader, when you see forward little Johnnies about—especially at railway stations. Such places are the chief resort of this class of pickpockets, and we hear that theatres and churches, just as the people are coming out, are favourite haunts—the women creating a stoppage at the door, and the children taking advantage of it. Women’s pockets are much more easily picked than men’s, for the reason that the opening through the dress to it is larger, and it hangs by its weight free of the person. In a crowd, the operation is easy enough, as the general pressure masks the movement of the depredator’s hand; when the victim is walking, a more artistic management is required. The hand is inserted at the moment that the right leg is thrown forward, because the pocket then hangs behind the limb, an essential condition for the thief, as the slightest motion is otherwise felt upon the leg. The trowser-pockets of a man are never attempted in the streets: but in a crowd, as at a race, he can be cleaned out by a school of mobsmen of everything in his possession, with little fear of detection. The first step is to select their victim; to do this demands some caution; and if they cannot see whether he carries a purse, and if they have no opportunity of watching him pull it out, they will feel all his pockets. The “spotter,” as he is called, passes his hand across the clothes seemingly in the most accidental manner; sometimes twice when he is in doubt. The fact that there is booty being ascertained, the confederates surround him, and wait for the coming-off of a race. Just as the horse is at the winning-post, there is a rush forward of the crowd: of this the mobsmen take advantage, while the victim, perhaps, for better security, keeps his hand over his pocket, but in vain. At a critical moment the man behind tips his hat over his eyes, instinctively he lifts up his hand to set it right, and the next moment his pocket is hanging inside out. Few betting men who attend much at races have escaped being thoroughly cleaned out. It is rarely that Londoners are robbed in the streets; they are too busy, and move on too fast. Country people form the chief game of the light-fingered gentry: as they stare about, they instantly betray themselves to their watchful enemy, and in the midst of their admiration at everything about them, fall an easy prey. The thief in
- 60. search of purses or handkerchiefs always makes his way trout-like against the stream. There are places, which, to carry out our piscatorial analogy, seem “ground-baited” for these fishers. Temple Bar, St. Paul’s Churchyard, the Shoreditch end of Bishopsgate, Holborn, Cheapside, and other crowded thoroughfares, all afford excellent sport for the pickpockets, and any one acquainted with their “manners and customs” may occasionally see them exercising their craft at these localities, if he watches narrowly. They look out for a temporary stoppage in the stream of people, and a horse fallen in the highway, an altercation between a cabman and his fare, a fight, a crowd round a picture-shop, are all excellent opportunities, of which they instantly take advantage. The May meetings at Exeter Hall, however, form the most splendid harvests for the pickpocket. If the members of the various religious denominations who flock thither escape the hustle on the hall stairs, they are waited upon with due attention in the omnibus. Ladies and gentlemen who attend these May meetings are well known to be “omnibus people:” they lodge or visit, for the short period of their sojourn in town, either at Islington, Clapham, or Camberwell, and the “Waterloos” and the “Victorias” are followed by the fraternity as certainly as a sick ship in the tropics is followed by the sharks. Omnibuses are generally “worked” by a man and a woman; the woman seats herself on the right-hand side of the most respectable- looking female passenger she can see, and the man if possible takes a place opposite the individual to be operated upon. If she be a young person, the man “stares her out of countenance,” and, whilst confused by his impertinence, the “pal,” by the aid of a cloak thrown over her arm, or, if a man, by passing his hand through the pocket of his cloak made open on the inside for the purpose, is able to rifle her pockets at leisure. If the victim be a middle-aged or elderly lady, her attention is engaged in conversation whilst the clearing-out process is going on. The trick done, the confederates get out at the first convenient opportunity. It is very rarely that a pickpocket pursues his avocation alone; but a case has been reported lately in the newspapers, which proves that a clever artist can work single-
- 61. handed. A man named William Henry Barber was charged at the Worship-street court with robbing a lady of her portemonnaie in a Stoke Newington omnibus: he was well known to the police, but had generally escaped by his adroitness. His manœuvres were thus described by a lady, a resident of Stoke Newington, who had been robbed by him on a previous occasion:— “She had got into an omnibus,” she said, “at Kingsland, several weeks back, to convey her to town, and found herself next to a gentlemanly-looking stout man, who was dressed in sober black, with a white neckerchief, and apparently a dissenting minister. The gentleman gradually encroached upon her, and pressed upon her; but she thought nothing of it, as he was very intent upon reading a newspaper the whole way—so intent, indeed, that she did not see his face, and he did not seem to notice that his newspaper several times partially covered her dress. The stranger shortly afterwards got out, and she did so also in a few minutes, and upon then placing her hand in her pocket to make some purchase, she found that her purse had been stolen, and with it seven sovereigns and a quantity of silver.” The “Dissenting Minister” had evidently worked the Stoke Newington road regularly, and no doubt the “sober black” and the white handkerchief were assumed with a perfect knowledge of the “serious” class of passenger he was likely to encounter in omnibuses running to that suburb. Robberies of this kind have enormously increased of late. The security with which pickpockets can work, withdrawn as they are from the surveillance of the police, is a great incentive to thieves to take to this particular line of business. The earnings of what is called a “school” of boys, who pick pockets in concert, under the eye of a master, must be considerable; for we were shown, some time since, a bill made out by one of those Fagins for the board and lodging of his hopeful youths, from which it appeared that the regular charge for each was two guineas a week! This person was well known some years since on the Surrey side of the water as Mo Clarke. He attended races, dressed in the deepest
- 62. black, with his young assistants in jackets and turned-down collars; and the whole group, to the eye of the general observer, presented the sad spectacle of a widower left with a family of young children to lament the loss of an attached mother. Their appearance disarmed suspicion, and enabled them to empty the pockets of those around them at their leisure. The subsequent fate of two of the children, though nursed in hypocrisy and vice, proves that the old saying, “once a thief always a thief,” is not invariably correct, for they are at the present moment flourishing cab and omnibus proprietors. The advantage of working out of sight of the police has lately led some of the swell mob to go to church, prayer-book in hand, and pick pockets either in the pews or while the congregation is coming down the aisle. Women are the greatest adepts at this kind of thieving, and they are constant attendants at confirmations, plundering in sight of the most touching rite of the Church. The dress of these females is perfect enough; but with them, as with most other members of the swell mob, the finish is entirely on the outside; they scarcely ever have any education, and the moment they open their mouths they betray themselves. This fact is of especial service in detecting another large class, of thieves—the shoplifters. A lady cannot go into the shop of any silkmercer or linendraper without being struck with the rude manner in which the shopman clears the counter immediately the purchaser takes her seat. The plundering to which they are subjected is some excuse for their suspicions, for the assistants cannot tell at first who the customer may be, and if expensive goods were left exposed while their backs were turned, serious robberies would inevitably occur. The value of the manner of speech, as diagnostic of character, was exemplified not long since at Messrs. Swan and Edgar’s, where a lady-like person asked to look at some “wallenciens.” A watch was kept upon the “lady,” and she was speedily detected secreting a card of valuable lace. The extent of pilfering carried on even by ladies of rank and position is very great; there are persons possessing a mania of this kind so
- 63. well known among the shopkeeping community, that their addresses and descriptions are passed from hand to hand for mutual security. The attendants allow them to secrete what they like without seeming to observe them, and afterwards send a bill with the prices of the goods purloined to their houses. Jewellers’ shops are especially open to a class of thieving termed “palming.” One of the gang goes in first, and engages the attention of the assistant; then another drops in, and makes inquiries for some article which is on the other side of the shop; then perhaps a third, without recognizing his companions, follows, and asks for something, saying he is in a hurry, as he has to be off by a certain train, and at the same time pulls out his watch to show his eagerness to be served. The shopkeeper’s attention is thus diverted from the confederates, who rob the trays before them of their valuable contents. Some of these fellows are so dexterous that, if they perceive any person watching them, they can “palm” back the goods they have secreted, and, on being accused, put on an appearance of injured innocence, which makes the tradesman believe that his own eyes must have deceived him. The higher order of thieves will sometimes “ring the changes,” as it is called. This must be ranked among the fine arts of swindling. They will call on first-rate houses, and request to be shown valuable pieces of jewellery, such as diamonds, necklaces, and bracelets, which are kept in cases. Having noted the case, they go away, promising to call with “a lady.” A case exactly similar is then made, with which they call a second time, and ask to see the identical bracelet they before admired, and substituting the empty case for that containing the jewels, depart with an apparent inability to decide upon the purchase. Many robberies to a heavy amount have taken place in this manner. Jewellers are liable to be attacked from without as well as from within. From the narration communicated by a prisoner to Captain Chesterton, when governor of Coldbath-fields prison, we extract the following method of procedure in what is termed “starring the glaze:”— “One or two parties divert attention while another ‘stars.’ This is either done by a diamond, or by inserting a small penknife through
- 64. the putty, near the corner of a pane, and cracking it; the wet finger carries the crack in any direction; an angle is generally formed. The piece is wrought to and through, and then removed; if necessary, another piece is ‘starred’ to allow of the free ingress of the hand. In a retired neighbourhood an opportunity is taken of tying the door, in order to prevent any one coming out, and on passing of a heavy carriage the hand is driven through a square of glass, upon which has been laid a piece of strong paper, coated with treacle, to prevent noise from the glass falling, and then articles of value are removed. This is termed spanking the glaze. At other times the parties intending to star go a night or two before and break one of the lower squares of glass, a watch is then put upon the shop to know when the square is renewed, which, of course, the putty being soft, can be removed at pleasure; a piece of leather, upon which is spread some pitch, being applied to the square to prevent it falling when pushed in. Much time is saved this way.” We often hear of the march of intellect in thieving, and the height to which its professors have carried it in these latter days. There could be no greater delusion; all the tricks of card-sharpers, ring-droppers, purse-cutters, c., are centuries old, and it does not appear that they are performed a bit more adroitly now than in the days of Elizabeth. Mr. Charles Knight, in his charming paper on London rogueries, gives examples of the tricks of the Shakspearian era, which prove, as he observes, that pickpocketing in all its forms was taught as cleverly in the days of the Tudors as by Fagin and his boys in “Oliver Twist.” His account of a school of thieves discovered in 1585 is an instance:— “Among the rest they found one Wolton, a gentleman born, and sometimes a merchant of good credit, but fallen by time into decay. This man kept an alehouse at Smart’s Key, near Billingsgate, and after, for some misdemeanour, put down, he reared up a new trade of life; and in the same house he procured all the cut-purses in the city to repair to his house. There was a schoolhouse set up to learn young boys to cut purses. Two devices were hung up—one was a
- 65. pocket and another was a purse. The pocket had in it certain counters, and was hung about with hawk’s bells, and over the top did hang a little scaring bell; the purse had silver in it, and he that could take out a counter without any noise was allowed to be a public Foyster; and he that could take a piece of silver out of the purse without noise of any of the bells, was adjudged a judicial nypper, according to their terms of art.” The tricks we have enumerated all require cunning, lightness of hand, and address, rather than strength and courage. As the swellmobsman stands at the head of this school, so the cracksman or housebreaker stands on the highest pinnacle of the other great division of crime which attains its ends by force and courage. Since the ticket-of-leave system has been in action, this department has flourished to an alarming degree. The released convict re-enters the community with the enlarged experience of the hulks and with a brutal disregard of danger. Suddenly thrown upon his resources, with a blasted character, society leaves him no better means of livelihood than his old course of crime. One fellow who was brought up to Bow-street had committed no less than four burglaries within three weeks after he had been liberated! Bands of ruffians, with crape masks and with deadly arms, stand by the bed at dead of night, and, after robbing and terrifying their victims, leave them gagged and bound in a manner that would disgrace banditti. It is true these burglaries are confined to lonely houses situated in the country; but housebreaking has been on the increase of late even in the metropolis. Some of the craftsmen have become so expert, that no system of bolts or bars is capable of keeping them out. It may be as well to state, however, that a sheet of iron, on the inside of a panel, will often foil the most expert burglars; and all operators of this class who have opened their minds upon the subject to the prison authorities admit that it is totally impossible, without alarming the inmates, to force a window that is lightly barred with a thin iron bar and supplied with a bell. A shutter thus protected, and which gives a little with pressure, will not allow the centrebit to work without creating a motion which is sure to ring the alarum.
- 66. Most burglaries of any importance, especially those in which much plate is stolen, are what is termed “put up;” that is, the thieves are in correspondence with servants in the house, or with those that have been discarded. Many robberies that appear to have been accomplished in a most wonderful manner from without, are committed from within. In “put up” robberies, however, the thieves seldom allow the confederate in the house to know when the robbery is to come off, for fear of what is termed a “double plant;” that is, lest the person who originally “put up” the robbery should, from the stings of conscience, or for other reasons, have officers in waiting to apprehend them. It is quite sufficient for adroit burglars to know where the valuables are kept, and the general arrangements of the house. We are indebted to the Yankees for an extremely clever method of gaining entrance to hotel bed-chambers, even when the inmate has fastened the door. The end of the key which projects through the lock is seized by a pair of steel pliers, and the door is unlocked whilst the traveller sleeps in fancied security. Several robberies of this kind have lately taken place. The most ingenious pilfering of the “put up” kind we ever heard of occurred many years ago in a large town in Hampshire. A gang of first-rate cracksmen, having heard that a certain banker in a country town was in the habit of keeping large sums of money in the strong box of the banking-house in which he himself dwelt, determined to carry it off. For this purpose the most astute and respectable-looking middle- aged man of the gang was despatched to the town, to reconnoitre the premises and get an insight into the character of their victim. The banker, he ascertained, belonged to the sect of Primitive Methodists, and held what is termed “love-feasts.” The cracksman accordingly got himself up as a preacher, studied the peculiar method of holding forth in favour with the sect, wore a white neckerchief, assumed the nasal whine, and laid in a powerful stock of scripture phrases. Thus armed, he took occasion to hold forth, and that so “movingly,” that the rumour of his “discourses” soon came to the ears of the banker, and he was admitted as a guest. His foot once inside the doors, he rapidly “improved the occasion” in his own peculiar manner. The intimacy grew, and he was speedily on
- 67. such terms of friendship with every one in the house, that he came and went without notice. He acquainted himself with the position of the strong box, and took impressions in wax of the wards of the locks. These he sent up to his pals in town, and in due course was supplied with false keys. With these he opened the strong box, made exact notes of the value and nature of its contents, and replaced everything as he found it. A plan of the street, the house, and of the particular chamber in which the treasure was kept, was then prepared and forwarded to the confederates in London. He persuaded his kind friend the banker to hold a love-feast on the evening fixed for the final stroke. A few minutes before the time appointed for the robbery, he proposed that the whole assembly should join with him in raising their voices to the glory of the Lord. The cracksman laboured hard and long to keep up the hymn, and noise enough was made to cover the designs of less adroit confederates than his own. The pseudo-preacher, to disarm suspicion, remained with his friend for a fortnight after the theft, and on his departure all the women of the “persuasion” wept that so good a man should go away from among them! In a large number of cases the servants are only the unconscious instrument in the hands of the housebreaker. We will venture to say that more house robberies are committed through the vanity of servant girls than from any other cause. A smart young fellow, having heard that plunder is to be obtained in a certain house, manages to pick up an acquaintance with one of the female domestics, and makes violent love to her. We all know how communicative young women are to their sweethearts, and the consequence is, that in a short time he gets from her every particular that he requires,—the habits of the family, the times of their going out, the position of the plate-chest, and the fastenings of the doors. Where only a servant of all-work is kept, the process is more simple. The lover calls in the absence of the family at church, proposes a walk, and takes charge of the street-door key, which, unseen to the girl, is passed to a confederate; and whilst the polite lover and his lass are enjoying the cool of the evening the house is
- 68. being ransacked. An investigation took place at the Lambeth Police Court a few months ago, where the poor girl who had been made the tool of the housebreaker attempted to commit suicide in order to prevent the consequences of her folly. Her account of the manner in which the “plant” was made upon her, affords a good example of the style of “putting up” a house robbery:— “The young man with whom she had casually become acquainted called after the family had gone out, and she asked him into the back parlour. He then asked her to dress and go out with him, and he remained in the back parlour while she dressed. While in the back parlour he asked her if she could get a glass of wine, and she told him that she could not, as the wine was locked up. He said it did not matter, as they should have one when they went out, and that he expected to meet his sister at the Elephant and Castle. They then left the house and went for a walk, and on reaching the Elephant and Castle remained there for some time, waiting for the young man’s sister, but did not see her. They next proceeded to a public- house, where they had a glass of brandy-and-water, and the young man accompanied her to the end of the street, where they parted, with the intention that they should meet at one o’clock on the following day and spend the afternoon together. On going to unlock the door, she found it ajar, and on going in, found that the house had been robbed. On discovering this, she did not know what to do, but thought she would make up a story about thieves having got into the house, and took up the knife and chopped her hand; but after this, not knowing how to face her master or mistress after being so wicked, she took up the knife again, intending to kill herself, and inflicted the wound on her throat.” This confession was enough for the officers, and her “young man,” with his confederates, were caught and convicted. The frequency of these robberies should put housekeepers on their guard as to what followers are allowed, lest the “young man” should turn out to be a regular cracksman in disguise. We bid the housekeeper also beware of another danger that sometimes threatens him when he has an
- 69. empty house for a neighbour. Thieves always, if possible, make use of it as a basis of operations against the others. They creep towards the dusk of evening, when the inmates are generally down stairs, along the parapet, and enter successively the bedrooms of the adjoining tenements. As many as half a dozen houses have thus been robbed on the same occasion. Police-constables always keep a careful watch upon these untenanted houses, by placing private marks on some part of the premises; and if any of these signs are disturbed, they suspect that something is wrong, and make a further examination. In the City, where an immense amount of valuable property is stored in warehouses, the private marks are much more used than in other portions of the metropolis, and are continually changed, lest they should become known to thieves and be turned to their advantage. Professional beggars are almost without exception thieves; but as they are generally recruited from the lowest portion of the population, they never attain any of the higher ranks, but confine themselves to petty acts of filching, or to cunning methods of circumventing the honest. The half-naked wretch that appears to be addressing the basement floor in piteous terms, has a fine eye for the spoons he may see cleaning below; and the shipwrecked sailor just cast ashore from St. Giles’s would be an awkward person to meet with in a dark suburban lane. Professional beggars are migratory in their habits. They travel from town to town, not in the filthy rags we are accustomed to see them in, but in good clothing; the rags are carried by their women, and are only donned when they are nearing the place in which they intend to beg. There is an audacious class of thieves, termed “dragsmen,” who plunder vehicles. At the West End they chiefly operate upon cabs going to or coming from the railway stations. As this kind of thieving is carried on under the very eyes of the foot-passengers, it is rarely attempted except in the dusk of the evening. The dragsman manages to hang on behind, as though he were merely taking a surreptitious ride, but in reality to cut leather thongs and undo
- 70. fastenings, and be able at any convenient moment to slip off a box or parcel unobserved. The carelessness of the public is the best confederate of this sort of thief. In the case of Lady Ellesmere’s jewels, the box was put not inside, but outside, the cab in which the valet rode, and not in the middle of other boxes, but the hindermost of all—just the place in which the dragsman would have planted it. It is now known that the robbery was effected between Berkeley Square and Grosvenor Square, as a man was seen with the package standing at the corner of Mount Street, Davies Street, bargaining with a cabman to take him to the City. The man and his booty were driven to a public-house, but the box must have been shifted immediately, for in two hours from the time it was lost it was found rifled of its contents in a waste piece of ground in Shoreditch. It might perhaps for a moment be suspected that this was a “put up” robbery, but we are precluded from adopting this view of the case, as it is, we believe, suspected that the man sold the jewels, which were worth perhaps 25,000l., for a very trifling sum. He must have been entirely ignorant of their value, and having by a chance stroke obtained a magnificent booty, threw it away for an old song. Not many weeks after this extraordinary robbery, a plate-chest of her Majesty was stolen from a van between Buckingham Palace and the Great Western Railway. There were persons walking alongside the vehicle, and it seems marvellous how it could be possible to remove unseen a heavy chest under such conditions; but every facility was given in this case, as in the former, for the plunderers to do their work unmolested. In the first place the box was put in such a position that its bottom came flush with the ledge of the van. Next, the journey from Buckingham Palace to Paddington was, in the driver’s idea, too far to go without baiting on the way; therefore bait he did at a little public-house, and every person in charge of the property went inside to drink. According to their own account, they did not stop more than a minute; this minute was enough: like Laertes, the thief might have said, “’Twill serve.” In this instance also the box was found empty in a field at Shoreditch, and it is believed that a ticket-of-leave man had a hand in both robberies.
- 71. The habits of thieves have been somewhat modified since the institution of the new police, and the adoption of the principle of prevention instead of detection, in dealing with the criminal population. In the time of the old Bow-street Runners the different classes of thieves had their houses of call, in which they regularly assembled. The arrangement was winked at by the magistrates, and approved by the officers, as useful to them in looking after offenders that were wanted. John Townsend, when speaking of the supposed advantage of these flash houses, said, “I know five-and-twenty, or six-and-twenty years ago, there were four houses where we could pop in, and I have taken three or four, or five or six of them at a time, and three or four of them have been convicted, and yet the public-house was tolerably well conducted too.” Perhaps officers who lived upon the capture of thieves had good reason for maintaining these flash houses, in which most robberies were concocted; the case is far different now that the police are paid by day rather than by piece-work, by weekly salary rather than by blood-money, and all known flash houses have long been discontinued. Some fifteen years since a few remained in the Borough, but Superintendent Haynes broke them up, and rooted them out. Thieves cannot meet now in respectable houses, for if they did, the constables would become aware of the fact, and the landlord would speedily lose his license. The passing of the Common Lodging-house Act has also assisted in dispersing the desperate gangs, one of which, known under the name of “The Forty Thieves,” infested the town a few years since. It may be asked, what sort of mutual fellowship exists among these outcasts who live below the surface of “society”? Of the seven or eight thousand thieves in the metropolis, very few are acquainted with each other; they are, in fact, divided into as many sections as are to be found among honest men. Beyond their own peculiar set they do not associate with their kind. The swell-mobsman is as distinct a being from the cracksman as a Bond-street dandy from a South-Sea islander; they do not even talk the same slang, and could no more practise each other’s art, than a shoemaker could make a table. These natural divisions of the underground world of rogues immensely facilitate the operations of the police. The manner in
- 72. which they do their work is also in some cases a pretty good guide to the detectives. Skill and individuality is evinced in unlawful as well as in lawful pursuits—in the manner in which a door is forced, as much as in the style a picture is painted; and a clever officer, after carefully examining a door or a window, will sometimes say, “This looks like ‘Whiteheaded Bob’s work,’” or “‘Billy-go-Fast,’ must have had a hand in this job.” The leading swell-mobsmen are the only class of thieves who “touch,” if we may term it, the ordinary society of better men. The practitioner in this line must dress and be as much like a gentleman as possible, in order to pursue his avocation without suspicion. Accordingly, he lives with a woman, who passes for his wife, in genteel lodgings, and generally in the drawing-room floor. As his earnings are often very large, he has everything about him of the most expensive kind; his style of living is luxurious, and he drinks nothing less than hock and champagne. He sometimes keeps a banking account, and one man named Brown, lately apprehended, had a balance at his banker’s of 800l.! As the members of this fraternity work wholly in the daytime, going out in the morning and returning in the evening, the landlady believes that they are engaged in mercantile pursuits, and have business in the City; and, as it is part of their game to pay their way liberally, she esteems them to be model lodgers! The domestic habits of thieves are all pretty much alike; fluctuating between the prison and the hulks, they exhibit the usual characteristics of men engaged in dangerous enterprises. They mainly pass their time, when not at “work,” in gambling, smoking, and drinking, and in listening to the adventures of their companions. It must be remembered, however, that the professed thief, even if he drinks, is never drunk; he is employed in desperate undertakings which require him to have his wits about him quite as much, if not more than the honest man. When a pickpocket is flush of money, he spends it in the most lavish manner,—takes a tour with his female companion to the Isle of Wight, or to any other place he has a wish
- 73. to see, and puts up at the best hotels. In some of these trips he thinks nothing of spending 30l. in a fortnight, and when the money is gone he comes back again “to work.” Thieves are generally faithful to each other; indeed the community of danger in which they live develops this virtue to an unusual extent. If a “pal” is apprehended, they cheerfully put down their guinea apiece to provide him with counsel for his trial; and if he should be imprisoned, they make a collection for him when he comes out. A curious circumstance is the rapidity with which news of any of the body having been arrested travels among his companions. We are assured that no sooner is a young thief captured and taken to the station-house, although he may have been plundering far away from his home, than some associate brings him his dinner or tea, as a matter of course. The best class of swell-mobsmen sometimes act upon the joint-stock principle “with limited liabilities.” When a good thing is in prospect—a gold-dust robbery or a bank robbery—it is not unusual for several of them to “post” as much as 50l. apiece in order to provide the sinews of war to carry on the plan in a business-like manner. If in the end the job succeeds, the money advanced is carefully paid back to the persons advancing it—several of whom have lived for years on plunder thus obtained, without the police being able to detect them. Often the receivers make these adventures in crime, and plot the robbery of a jeweller’s shop with as much coolness and shrewdness as though it were an ordinary mercantile speculation, and the produce is disposed of in the same business-like manner. Watches are what is termed “re-christened,” that is, the maker’s names and numbers are taken out and fresh ones put in; they are then exported in large quantities to America. All articles of plate are immediately thrown into the crucible and melted down, so as to place them beyond the hope of identification. In many cases, when the receiver cannot thoroughly depend upon the thief, it is, we believe, customary to employ intermediate receivers so as to render it impossible to trace the property to its ultimate destination. It must not be supposed that the passion for gain is always the sole incentive to robbery. “Oh, how I do love thieving! If I had
- 74. thousands, I’d still be thief;” such were the words uttered by a youth in Coldbath-fields Prison, and overheard by the governor.[49] If the machinery for preventing and detecting crime has so vastly improved within this present century, the same may be said for the method of dispensing justice. Up to as late as 1792, the magistrates of Bow-street—the first “police-office,” as it was then termed—were paid in that most obnoxious of all modes, by fees, which were often obtained in a manner so disgraceful that the magistrates got the name of “trading justices” and “basket justices.” Our old friend John Townsend, whom we must summon once more to our aid, gives an insight into their proceedings, and he knew them well. He said, “The plan used to be to issue warrants, and to take up all the poor devils in the streets, and then there was the bailing them, 2s. 4d., which the magistrate had. In taking up a hundred girls, that would make, at 2s. 4d., 11l. 13s. 4d. They sent none to jail, for the bailing them was so much better!” The old Bow-street worthy then draws a picture of the magistrate settling the amount of these ill-gotten fees with his clerk on the Monday morning. The “basket justices” were so called, because they allowed themselves to be bought over by presents of baskets of game. These enormities were so glaring, that, according to Townsend, “they at last led to the Police Bill, and it was a great blessing to the public to do away with these men, for they were nothing better than the encouragers of blacklegs, vice, and plunderers. There is no doubt about it.” In 1792 seven other “offices” were established, namely, Queen-square, Great Marlborough-street, Hatton Garden, Worship-street, Lambeth, Shadwell, and Union-street, each office having three magistrates, who did the duties alternately. These, by the addition of the suburban courts, have since been augmented to eleven. They form the judgment-seats to which all offenders in this great capital of 2,500,000 inhabitants are brought, either to be punished summarily, or to be remanded to the sessions to take their trial. The police-courts may be likened to so many shafts sunk in the smooth surface of society, through which the seething mass of
- 75. debauchery, violence, and crime, are daily bubbling up before the public eye. A spectator cannot sit beside the magistrate on the bench for a couple of hours without feeling that there are currents of wickedness flowing among the population as fixedly as the trade- winds in the tropics. A panorama of sin passes before his eye which he shudders to think is only like a single thread drawn from the fabric of vice which underlies the whole system of elegant, punctilious, and accomplished metropolitan life. On every case that comes before him the magistrate unassisted has to decide rapidly and justly, unless he desires to call down upon his head the thunders of an ever-watchful press. In addition to his judicial duties, he has to answer numberless questions, and to give advice upon law points to distressed persons: and all this amid a pestilential atmosphere which is calculated to depress both body and mind. Nevertheless, the work is done admirably, and justice, as speedy as that dispensed by cadis in Eastern tales, and much more impartial, is dealt to the throng brought before him. From an analysis of the Criminal Returns of the Metropolitan Police, it is apparent that crimes have their peculiar seasons. Thus attempts to commit suicide generally occur in the months of June, July, and August, and rarely in November, according to the commonly accepted notion; comfort, it is evident, is considered even in the accomplishment of this desperate act. Common assaults and drunkenness also multiply wonderfully in the dog-days. In the winter, on the contrary, burglaries increase, and, for some unknown reason, the uttering of counterfeit coin. The character of the cases brought before the police-courts varies, in some degree, according to the neighbourhood and other causes. Bow-street still maintains the pre-eminence over the other courts which it exercised in the old days, when the horse-patrol and the detective police, known as the Bow-street runners, were in existence; and this it does in consequence of its special jurisdiction over persons who are amenable to foreign law. The cases of this class—arson, murder, or bankruptcy—are heard in private, generally
- 76. by the chief magistrate, and the depositions are forwarded direct to the Foreign Office. Ticket-of-leave men who have committed fresh offences, are here deprived of their tickets and apprehended by a warrant from the Home-Office. All Inland Revenue and Post-Office cases, such as stealing from letters, are adjudicated upon exclusively at Bow-street, which is, in fact, the Government office. The Thames police deals with mutinies and murders committed on the high seas, and all disputes under the Mercantile Marine Act come as a matter of course to this court, together with the major portion of the criminals, the scene of whose offences is in the docks and on the river. Drunkenness, the vice of the sailors, and the insubordination arising out of it, form a very large portion of the charges of the district. Worship-street is famous, or rather infamous, for wife-beaters. The reason is curious, and supplies a hint to philanthropists to reform the dwellings of the poor, rather than pass harsh acts of parliament against the husbands, which in many cases only serve to aggravate the evils arising from their brutality. The majority of the wife-beaters come from Bethnal-green, where there are a great number of large old mansions let out to the working- classes in floors or flats. Sometimes as many as twenty families live in the same house. The children play about in the passages as a neutral ground, disputes arise, and the mothers take the parts of their respective offspring with discordant fierceness. This drives the men to the public-houses, where they drink their porter iced and listen to more pleasant sounds in the shape of gratuitous concerts. The wives in turn are driven to the tavern doors to seek their mates, with words not too conciliatory, and are brutally assaulted by the drunken husbands, who are taken up the next day and get six months’ imprisonment, the family being in most instances irretrievably broken up and ruined thereby. Some of the magistrates, seeing the baleful working of the system, have attempted a solution of the difficulty by making the husband promise to allow the wife to receive his weekly wages from his master, whose consent to the arrangement has been given. In many instances this plan has worked well, since the husband knows that on the slightest
- 77. infringement of the agreement his spouse may give him six months’ imprisonment, judgment in the case having been only suspended. But this power, again, is often abused by the woman, and it is a common thing for them on the least threat of their mates to say, “Mind what you are about, or I will give you ‘a sixer.’” Cases of begging are principally heard at the Marlborough-street police-court, as the rich streets in its neighbourhood are the main scenes of the nuisance. Blind beggars especially affect Regent- street, Oxford-street, and Piccadilly, the most thronged thoroughfares in the West End. We warn our readers against their charitable tendencies for these people. If the truth was known, the cry, “Pity the poor blind!” far from exciting their pity, would arouse their disgust. Blind beggars, as a class, are the most profligate scoundrels in the metropolis, thinking of nothing but their grosser appetites, and plundering the charitable for their satisfaction. One of these men lately taken into custody was discovered seated at the breakfast-table with ham and fourteen poached eggs before him! At the Westminster police-court the foot-guards are continually visitors against their will; but it is remarked as extraordinary that not one of the horse-guards has been charged here for years. A custom has grown up of making the police magistrates the almoners of the public in cases which have attracted the attention of the charitable through the medium of the press. Many a poor forsaken creature has suddenly found himself not only famous, but comparatively rich, by the simple process of telling his tale in one of these courts. The news of it flies through the country in the pages of the Times, and in the course of two or three mornings the magistrate is oppressed with post-office orders for the benefit of the sufferer, the donors simply requesting that their gifts should be acknowledged in the public journals. The annual receipts at the different courts for special cases must amount to a large sum; and there is in addition a constant flow of small sums towards the poor- box, the contents of which are distributed at the discretion of the magistrate. The annual income from this latter source is about 300l.
- 78. per annum at Marlborough-street, and at Bow-street respectively, the greater portion of which is given to deserving objects whose cases have come before the court, and the remainder is dispensed at Christmas to the poor of the neighbourhood in the shape of coals and candles. We are particularly anxious to make this fact known, in order that the charitable may be aware that their gifts are well bestowed. The magistrates do not, we believe, encourage these donations, as they consider that the distribution of alms is incompatible with their office; but, on the other hand, it cannot be denied that a vast amount of temporary aid is thus given to persons whose needs cannot be satisfied by the union workhouse. Deserving people are often furnished with the means of obtaining a livelihood, workmen whose tools have been burned in a conflagration supplied with new ones, and in some cases women left behind by their husbands, under circumstances of peculiar hardship, have been provided with a passage to Australia. The thousands in England who only want to know where genuine misfortune exists to hasten to its relief, have a greater guarantee that they will not be imposed upon by these cases at the police-courts than by private solicitations, as the magistrates have the means of sifting the statements of applicants. Nevertheless, even these astute public servants are now and then deceived, and comparatively large sums have been received by them for persons who have afterwards been ascertained to be unworthy of relief; and in instances where the discovery took place in time, the money, by the direction of the donors, has been transferred to truer objects of charity. The fees, penalties, and forfeitures received at the eleven metropolitan police-courts and by the justices of the exterior police districts are very considerable; in 1855 they amounted to 11,315l. 16s. 6d. This sum goes towards defraying the expenses of the courts, which, together with the salaries of the officers, and other items, amounted in the same year to 63,021l. 0s. 5d. The expenditure may be considered reasonable, when it is remembered that 60,000 cases are annually disposed of, many of which require a minute knowledge of statute and of common law. The chief
- 79. improvement required is the improvement of the buildings. The Thames police-court is the only one at all suitable for its purpose. An enclosed yard is attached to it, in which the police-van can draw up and discharge its prisoners without exposing them to the public gaze, an important point in times of public excitement. Clerkenwell and Westminster are the next best-arranged courts, but both want space and air; Lambeth, though lately built, is a complete failure; many of the other courts are held in small private houses; and in those of Marlborough Street and Hammersmith, the business is transacted up stairs. In the latter court it is a common thing to hear it said of persons who have been taken before the magistrates—“he has been up the forty steps.” With the common people, with whom these institutions have mainly to deal, justice should be dispensed with regard to appearances; there should be the formality of the superior courts, and somewhat of their show. A magistrate sitting in a plain black dress like an ordinary gentleman, and a lawyer dispensing justice in his wig and gown, are two very different things to the lower classes, whatever they may be to educated persons; and the want of all official costume, and the huddled style of doing business, inseparable from the present confined space, is not calculated to inspire the people with much respect. The police should at least be put upon a level with the county-courts. The latter have to deal with less momentous interests. Questions of paltry debt cannot be put in comparison with questions involving the liberty of the subject; the power of committing to prison for six months with hard labour is far more important than that of adjudicating in money disputes under five pounds. It is not enough that justice is administered; it is the opinion which the people have of it that produces the effect, and until the judgment-seat is rendered dignified, and those who sit on it are clothed with the habiliments which distinguish the magistrate from the man, the law, by losing most of its impressiveness, will lose its moral power over delinquents. The vulgar terror of punishment may remain, but the lesson which is conveyed to the feelings by the solemn stateliness of the tribunal is entirely gone.
- 80. Welcome to our website – the perfect destination for book lovers and knowledge seekers. We believe that every book holds a new world, offering opportunities for learning, discovery, and personal growth. That’s why we are dedicated to bringing you a diverse collection of books, ranging from classic literature and specialized publications to self-development guides and children's books. More than just a book-buying platform, we strive to be a bridge connecting you with timeless cultural and intellectual values. With an elegant, user-friendly interface and a smart search system, you can quickly find the books that best suit your interests. Additionally, our special promotions and home delivery services help you save time and fully enjoy the joy of reading. Join us on a journey of knowledge exploration, passion nurturing, and personal growth every day! ebookbell.com