![3
Shree Ram Singh and Vincenzo Coppola (eds.), Mouse Genetics: Methods and Protocols, Methods in Molecular Biology,
vol. 1194, DOI 10.1007/978-1-4939-1215-5_1, © Springer Science+Business Media New York 2014
Chapter 1
Development of Pronuclear Injection-Based Targeted
Transgenesis in Mice Through Cre–loxP Site-Specific
Recombination
Masato Ohtsuka
Abstract
Microinjection of DNA into the pronuclei of zygotes is the simplest and most widely used method for
generating transgenic (Tg) mice. However, it is always associated with random integration of multiple
copies of the transgene, resulting in unstable, low, or no transgene expression due to positional effects
and/or repeat-induced gene silencing. In addition, random integration sometimes disrupts an endoge-
nous gene that can affect the phenotypes of Tg mice. Our recently developed pronuclear injection-based
targeted transgenesis (PITT) method enables the integration of a single-copy transgene into a predeter-
mined genomic locus through Cre–loxP site-specific recombination. The PITT method enables stable and
reliable transgene expression in Tg mice and is also applicable for generating knockdown mice. Therefore,
the PITT method could represent next-generation transgenesis that overcomes the pitfalls of conventional
transgenesis.
Key words Transgenic mouse, PITT, Pronuclear injection, Targeted transgenesis, Transgene expression,
Cre–loxP, Rosa26, Knockdown mouse
1 Introduction
Transgenic (Tg) mice have been widely used for analyzing gene
function and for creating animal models of human disease. The
most commonly used method to generate Tg mice is microinjec-
tion of a DNA solution into the pronuclei of fertilized eggs [pronu-
clear injection (PI)]. Although this is the simplest method and has
been utilized for more than three decades since Gordon et al. [1]
first generated Tg mice, the PI method has some limitations such as
an inability to control the transgene integration site and copy num-
ber. These drawbacks often lead to unexpected and unstable trans-
gene expression in Tg mice, probably due to positional effects
and/or the repeat-induced gene silencing [2–4]. In addition,
random transgene integration sometimes results in disruption of
an endogenous gene, which may cause unexpected phenotypes.](https://image.slidesharecdn.com/82813-250404180808-1d4463c7/85/Mouse-Genetics-Methods-and-Protocols-1st-Edition-Shree-Ram-Singh-23-320.jpg)
![4
Therefore, production and analysis of multiple Tg mouse lines are
always required for obtaining reliable results. Such a task is labori-
ous, costly, and time consuming. Although embryonic stem (ES)
cell-mediated targeted integration of transgenes can overcome
these drawbacks, it is more costly and time consuming than PI-based
transgenesis.
Recently, we developed a novel targeted transgenesis method
that enables the targeted integration of a single-copy transgene
into a predetermined genomic locus through simple PI [5]. We
have termed this technology as PI-based targeted transgenesis
(PITT). This method is based on Cre–loxP site-specific recombina-
tion. Cre (causes recombination) is an enzyme derived from bacte-
riophage P1 that binds to a 34 bp region of loxP [locus of crossing
over (x), P1] and catalyzes recombination between two loxP
sequences. The loxP sequence consists of an 8-bp spacer region,
which determines direction, between two perfect 13-bp inverted
repeats. The Cre–loxP system has been widely used as a tool for
modifying the genome of many model organisms, including mice.
One of the most frequently utilized applications of the Cre–loxP
system is the excision of the floxed DNA fragment from the
genome of conditional knockout mice. In contrast, the PITT
method utilizes the Cre–loxP system for insertion of the DNA of
interest (DOI) into the genome (Fig. 1). For this purpose, we use
the mutant loxP sequences JT15 [left element (LE)] and JTZ17
[right element (RE)] in the inverted-repeat region [6] because
LE/RE double-mutated loxP, produced as a result of insertion
through JT15/JTZ17 recombination, is not recognized by the
Cre enzyme with high affinity, promoting the integration reaction
over the excision reaction. We also used the spacer-region mutant
lox2272 [7] in addition to JT15 and JTZ17 for efficient targeted
integration [8].
Almost all previous reports on Cre–loxP-based Tg mice pro-
duction have employed the Cre–loxP recombination reaction in ES
cells [9]. In this study, we aimed to develop a targeted transgenesis
method through Cre–loxP recombination in zygotes rather than
ES cells. To accomplish this, we first established seed mice lines
containing JT15 and lox2272 at the Rosa26 or the H2-Tw3 locus
by ES cell-based gene targeting [5]. These seed mice are main-
tained as homozygotes and their fertilized eggs are used for injec-
tion to generate targeted Tg mice. The donor vector containing
JTZ17 and lox2272 in addition to the DOI is co-injected into the
eggs along with a Cre expression plasmid or iCre (codon-improved
Cre recombinase) mRNA [10]. As a result, Cre–loxP recombina-
tion in zygotes may integrate the DOI into the tagged Rosa26 or
H2-Tw3 locus (Fig. 1). We applied the PITT method for the gen-
eration of a number of gene-overexpressing Tg mice such as vari-
ous fluorescent Tg mice [11]. In addition, we report the successful
knockdown of the tyrosinase gene using the PITT method [5].
Masato Ohtsuka](https://image.slidesharecdn.com/82813-250404180808-1d4463c7/85/Mouse-Genetics-Methods-and-Protocols-1st-Edition-Shree-Ram-Singh-24-320.jpg)
![5
Our previous data confirmed that the expression of transgene tends
to be highly reproducible and stable [11, 5]; therefore, only one
germline-transmissible line is sufficient for maintenance and analy-
sis. This is advantageous from the viewpoint of animal welfare
because it reduces the number of mice required. Therefore, PITT
could become widely used as a next-generation transgenesis
method in the near future.
2 Materials
1. Donor vector: The vector contains mutant loxP sequences,
such as JTZ17 and lox2272, in addition to the FRT sequence,
which is used for removal of extra sequences (Figs. 1 and 2).
2.1 Vector
Construction and DNA/
mRNA Preparation
Fig. 1 Schematic diagram of pronuclear injection-based targeted transgenesis (PITT). The donor vector is co-
injected with Cre expression plasmid or iCre mRNA into the pronuclei of fertilized eggs obtained from seed
mice. In case of injection with iCre mRNA, cytoplasmic injection and pronuclear injection are performed. As a
result of site-specific recombination between mutant loxPs after catalysis by the Cre enzyme, one copy of the
donor vector is integrated into the predetermined genomic locus (e.g., Rosa26 or H2-Tw3) and DOIex
mouse is
generated. Extra sequence containing vector sequence and selection marker (neo) is removed at the FLP–FRT
recombination step, resulting in the generation of the DOIΔex
Targeted Transgenesis Through Pronuclear Injection](https://image.slidesharecdn.com/82813-250404180808-1d4463c7/85/Mouse-Genetics-Methods-and-Protocols-1st-Edition-Shree-Ram-Singh-25-320.jpg)
Mouse Genetics Methods and Protocols 1st Edition Shree Ram Singh
- 1. Mouse Genetics Methods and Protocols 1st Edition Shree Ram Singh pdf download https://ebookfinal.com/download/mouse-genetics-methods-and- protocols-1st-edition-shree-ram-singh/ Explore and download more ebooks or textbooks at ebookfinal.com
- 2. Here are some recommended products for you. Click the link to download, or explore more at ebookfinal Mouse Models of Cancer Methods and Protocols 1st Edition Robert Eferl https://ebookfinal.com/download/mouse-models-of-cancer-methods-and- protocols-1st-edition-robert-eferl/ Transgenic Mouse Methods and Protocols 2nd Edition Marten H. Hofker (Auth.) https://ebookfinal.com/download/transgenic-mouse-methods-and- protocols-2nd-edition-marten-h-hofker-auth/ Mouse Models of Allergic Disease Methods and Protocols 1st Edition Jennifer Skelton https://ebookfinal.com/download/mouse-models-of-allergic-disease- methods-and-protocols-1st-edition-jennifer-skelton/ Yeast Genetics Methods and Protocols 1st Edition Jeffrey S. Smith https://ebookfinal.com/download/yeast-genetics-methods-and- protocols-1st-edition-jeffrey-s-smith/
- 3. Reverse Chemical Genetics Methods and Protocols 1st Edition Osamu Ohara (Auth.) https://ebookfinal.com/download/reverse-chemical-genetics-methods-and- protocols-1st-edition-osamu-ohara-auth/ Mouse Models for Drug Discovery Methods and Protocols 2nd ed. 2016 Edition Gabriele Proetzel https://ebookfinal.com/download/mouse-models-for-drug-discovery- methods-and-protocols-2nd-ed-2016-edition-gabriele-proetzel/ Plant Reverse Genetics Methods and Protocols Methods in Molecular Biology 1st Edition. Edition Andy Pereira https://ebookfinal.com/download/plant-reverse-genetics-methods-and- protocols-methods-in-molecular-biology-1st-edition-edition-andy- pereira/ Biofuel Crops Production Physiology and Genetics Bharat P Singh https://ebookfinal.com/download/biofuel-crops-production-physiology- and-genetics-bharat-p-singh/ Genetic resources chromosome engineering and crop improvement Vegetable crops 1st Edition Ram J. Singh https://ebookfinal.com/download/genetic-resources-chromosome- engineering-and-crop-improvement-vegetable-crops-1st-edition-ram-j- singh/
- 5. Mouse Genetics Methods and Protocols 1st Edition Shree Ram Singh Digital Instant Download Author(s): Shree RamSingh, Vincenzo Coppola (eds.) ISBN(s): 9781493912148, 1493912143 Edition: 1 File Details: PDF, 10.25 MB Year: 2014 Language: english
- 6. Mouse Genetics Shree Ram Singh Vincenzo Coppola Editors Methods and Protocols Methods in Molecular Biology 1194
- 7. ME T H O D S I N MO L E C U L A R BI O LO G Y Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651
- 9. Mouse Genetics Methods and Protocols Edited by Shree Ram Singh Stem Cell Regulation and Animal Aging Section, Basic Research Laboratory, NationalCancerInstitute,NationalInstitutesofHealth,Frederick,MD,USA Vincenzo Coppola GeneticallyEngineeredMouseModelingCore/DepartmentofMolecularVirology, ImmunologyandMedicalGenetics,WexnerMedicalCenter,ComprehensiveCancerCenter, TheOhioStateUniversity,Columbus,OH,USA
- 10. ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-4939-1214-8 ISBN 978-1-4939-1215-5 (eBook) DOI 10.1007/978-1-4939-1215-5 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2014942721 © Springer Science+Business Media New York 2014 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Editors Shree Ram Singh Stem Cell Regulation and Animal Aging Section, Basic Research Laboratory National Cancer Institute, National Institutes of Health Frederick, MD, USA Vincenzo Coppola Genetically Engineered Mouse Modeling Core/Department of Molecular Virology, Immunology and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center The Ohio State University Columbus, OH, USA
- 11. v Although evolution has separated mice and humans about 75 million years ago, they still share an incredible level of anatomical, physiological, and especially genomic resemblance. Modeling human disease in mice offers numerous advantages over other mammalian ani- mal models because they are small, easy to breed, available in large number of inbred strains, and their genome is fully sequenced. Most of all, early stage mouse embryos are conducive to in vitro manipulation, and many embryonic stem (ES) cell lines have shown robust homologous recombination and germline transmission in existence. All these ele- ments have allowed researchers to develop innovative technologies that efficiently edit the mouse genome in vivo. It is now possible to engineer targeted alterations such as gene knockout and knockin or elegant conditional gene modification, which allows temporal- spatial regulation and cell lineage tracing. Other genetic technologies such as Recombinase- Mediated Cassette Exchange (RMCE), which can generate allelic series of mutants and mutagenesis, can also be obtained by use of transposons. Over the years, almost all human diseases including cancer, diabetes, obesity, cystic fibrosis, arthritis, and heart and neurode- generative diseases have been modeled in mice. Mouse Genetics: Methods and Protocols provides selected mouse genetic techniques and their application in modeling varieties of human diseases. The chapters are mainly focused on the generation of different transgenic mice to accomplish the manipulation of genes of interest, tracing cell lineages, and modeling human diseases. Composed in the highly suc- cessful Methods in Molecular Biology series format, each chapter contains a brief introduc- tion, a list of necessary materials, systematic methods, and a notes section, which shares tips on troubleshooting to avoid known pitfalls. We hope that Mouse Genetics: Methods and Protocols would provide fundamental tech- niques and protocols to geneticists, molecular biologists, cell and developmental biologists, students, and postdoctoral fellows working in the various disciplines of mouse biology and modeling human disease. We would like to thank Prof. John M. Walker and the staff at Springer for their invita- tion, editorial guidance, and assistance throughout the preparation of the book for publica- tion. We also would like to express our sincere appreciation and gratitude to the contributors for sharing their precious laboratory expertise with the mouse community. Finally yet importantly, we would like to thank our family members for their continued support. Frederick, MD, USA Shree Ram Singh, Ph.D. Columbus, OH, USA Vincenzo Coppola, M.D. Preface
- 13. vii Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi PART I MOUSE GENETICS TECHNIQUES 1 Development of Pronuclear Injection-Based Targeted Transgenesis in Mice Through Cre–loxP Site-Specific Recombination . . . . . . . . . . . . . . . . . 3 Masato Ohtsuka 2 Generation of Conditional Knockout Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Kazuhito Sakamoto, Channabasavaiah B. Gurumurthy, and Kay-Uwe Wagner 3 Generating Mouse Lines for Lineage Tracing and Knockout Studies . . . . . . . . 37 Petra Kraus, V. Sivakamasundari, Xing Xing, and Thomas Lufkin 4 Generation of an Allelic Series of Knock-In Mice Using Recombinase-Mediated Cassette Exchange (RMCE). . . . . . . . . . . . . . . . . . . . 63 Anton J.M. Roebroek and Bart Van Gool 5 Generating Chimeric Mice from Embryonic Stem Cells via Vial Coculturing or Hypertonic Microinjection . . . . . . . . . . . . . . . . . . . . . 77 Kun-Hsiung Lee 6 Genetic Inducible Fate Mapping in Adult Mice Using Tamoxifen-Dependent Cre Recombinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Susanne Feil, Jana Krauss, Martin Thunemann, and Robert Feil 7 Intracytoplasmic Sperm Injection (ICSI)-Mediated Transgenesis in Mice. . . . . 141 Pedro N. Moreira and Lluís Montoliu 8 Generation of Bacterial Artificial Chromosome (BAC) Transgenic Mice . . . . . 157 Jane Beil and Thorsten Buch 9 Generation of Genetically Engineered Mice by the piggyBac Transposon System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Sheng Ding, Tian Xu, and Xiaohui Wu 10 Generation and Applications of MADM-Based Mouse Genetic Mosaic System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 Hui Zong 11 Generation of Mouse Lines Conditionally Over-expressing MicroRNA Using the Rosa26-Lox-Stop-Lox System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Claudia Piovan, Foued Amari, Francesca Lovat, Qun Chen, and Vincenzo Coppola 12 In Situ Hybridization (Both Radioactive and Nonradioactive) and Spatiotemporal Gene Expression Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 225 Olga Simmons, Esther M. Bolanis, Jian Wang, and Simon J. Conway Contents
- 14. viii PART II MOUSE GENETICS IN STEM CELLS 13 Isolation and Handling of Mouse Embryonic Fibroblasts . . . . . . . . . . . . . . . . 247 Kanika Jain, Paul J. Verma, and Jun Liu 14 Generation of Induced Pluripotent Stem Cells from Mouse Adipose Tissue. . . 253 Pollyanna Agnes Goh and Paul J. Verma 15 Pdx1 (GFP/w) Mice for Isolation, Characterization, and Differentiation of Pancreatic Progenitor Cells. . . . . . . . . . . . . . . . . . . . . . 271 Michael D. Williams, Wilson Wong, Amanda Rixon, Sarang N. Satoor, Anandwardhan A. Hardikar, and Mugdha V. Joglekar 16 Generation of Transgenic Mouse Fluorescent Reporter Lines for Studying Hematopoietic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 Andrei M. Vacaru, Joseph Vitale, Johnathan Nieves, and Margaret H. Baron 17 Analyzing Gene Function in Adult Long-Term Hematopoietic Stem Cells Using the Interferon Inducible Mx1-Cre Mouse System . . . . . . . . 313 Kristbjorn Orri Gudmundsson, Kevin Oakley, Yufen Han, and Yang Du 18 Generation of Transgenic Mice by Exploiting Spermatogonial Stem Cells In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Lalit Sehgal, Abul Usmani, Sorab N. Dalal, and Subeer S. Majumdar PART III MOUSE GENETICS IN MODELING HUMAN DISEASE 19 Methods for the Detection of Genome Instability Derived from Replication Stress in Primary Mouse Embryonic Fibroblasts . . . . . . . . . . 341 Spencer W. Luebben, Naoko Shima, and Tsuyoshi Kawabata 20 Transgenic Nude Mice Ubiquitously Expressing Fluorescent Proteins for Color-Coded Imaging of the Tumor Microenvironment. . . . . . . . 353 Robert M. Hoffman 21 Genetically Engineered Insertional Mutagenesis in Mice to Model Cancer: Sleeping Beauty. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367 Viive M. Howell and Emily K. Colvin 22 Generating Double Knockout Mice to Model Genetic Intervention for Diabetic Cardiomyopathy in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 Vishalakshi Chavali, Shyam Sundar Nandi, Shree Ram Singh, and Paras Kumar Mishra 23 Experimental Osteoarthritis Models in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . 401 Julia Lorenz and Susanne Grässel 24 Mouse Models and Methods for Studying Human Disease, Acute Kidney Injury (AKI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421 Ganesan Ramesh and Punithavathi Ranganathan Contents
- 15. ix 25 Mouse Models of Acute and Chronic Colitis . . . . . . . . . . . . . . . . . . . . . . . . . . 437 Santhakumar Manicassamy and Indumathi Manoharan 26 Manipulation and Assessment of Gut Microbiome for Metabolic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449 Sarang N. Satoor, Deepak P. Patil, Holly D. Kristensen, Mugdha V. Joglekar, Yogesh Shouche, and Anandwardhan A. Hardikar Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471 Contents
- 17. xi FOUED AMARI • Genetically Engineered Mouse Modeling Core/Department of Molecular Virology, Immunology and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA MARGARET H. BARON, M.D., PH.D. • Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA; The Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA JANE BEIL • Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München, Munich, Germany ESTHER M. BOLANIS • HB Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA THORSTEN BUCH • Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany VISHALAKSHI CHAVALI • Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, USA QUN CHEN • Genetically Engineered Mouse Modeling Core/Department of Molecular Virology, Immunology and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA EMILY K. COLVIN • Bill Walsh Translational Cancer Research Laboratory, Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney, St. Leonards, NSW, Australia SIMON J. CONWAY • HB Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA VINCENZO COPPOLA • Genetically Engineered Mouse Modeling Core/Department of Molecular Virology, Immunology and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA SORAB N. DALAL • Advanced Centre for Treatment Research Education and Cancer, Tata Memorial Centre, Navi Mumbai, India SHENG DING • State Key Laboratory of Genetic Engineering and Institute of Developmental Biology and Molecular Medicine, Fudan-Yale Biomedical Research Center, School of Life Sciences, Fudan University, Shanghai, China; Department of Genetics, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA YANG DU • Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, MD, USA ROBERT FEIL • Interfakultäres Institut für Biochemie, Universität Tübingen, Tübingen, Germany SUSANNE FEIL • Interfakultäres Institut für Biochemie, Universität Tübingen, Tübingen, Germany Contributors
- 18. xii POLLYANNA AGNES GOH • Research Department of Haematology, UCL Cancer Institute, University College London, London, UK BART VAN GOOL • Laboratory for Experimental Mouse Genetics, Center for Human Genetics, KU Leuven, Leuven, Belgium SUSANNE GRÄSSEL, PH.D. • Orthopaedic Surgery, Experimental Orthopaedics, Centre for Medical Biotechnology, University of Regensburg, Regensburg, Germany KRISTBJORN ORRI GUDMUNDSSON • Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, MD, USA CHANNABASAVAIAH B. GURUMURTHY • Department of Genetics, Cell Biology, & Anatomy, University of Nebraska Medical Center, Omaha, NE, USA YUFEN HAN • Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, MD, USA ANANDWARDHAN A. HARDIKAR • NHMRC Clinical Trials Centre, The University of Sydney, Camperdown, NSW, Australia ROBERT M. HOFFMAN • AntiCancer, Inc., San Diego, CA, USA; Department of Surgery, UCSD Medical Center, San Diego, CA, USA VIIVE M. HOWELL • Bill Walsh Translational Cancer Research Laboratory, Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney, St. Leonards, NSW, Australia KANIKA JAIN • Stem Cells and Reprogramming Group, Biological Engineering, Faculty of Engineering, Monash University, Clayton, VIC, Australia MUGDHA V. JOGLEKAR, PH.D. • NHMRC Clinical Trials Centre, The University of Sydney, Camperdown, NSW, Australia TSUYOSHI KAWABATA • Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA PETRA KRAUS • Department of Biology, Clarkson University, Potsdam, USA JANA KRAUSS • Interfakultäres Institut für Biochemie, Universität Tübingen, Tübingen, Germany HOLLY D. KRISTENSEN • NHMRC Clinical Trials Centre, The University of Sydney, Camperdown, NSW, Australia KUN-HSIUNG LEE • Division of Biotechnology, Animal Technology Institute Taiwan, Chunan, Miaoli, Taiwan JUN LIU • Stem Cells and Reprogramming Group, Biological Engineering Laboratories, Faculty of Engineering, Monash University, Clayton, VIC, Australia JULIA LORENZ • Orthopaedic Surgery, Experimental Orthopaedics, Centre for Medical Biotechnology, University of Regensburg, Regensburg, Germany FRANCESCA LOVAT • Department of Molecular Virology, Immunology and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA SPENCER W. LUEBBEN • Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA THOMAS LUFKIN • Department of Biology, Clarkson University, Potsdam, USA SUBEER S. MAJUMDAR • National Institute of Immunology, New Delhi, India SANTHAKUMAR MANICASSAMY, PH.D. • Department of Medicine and Cancer Center, Georgia Regents University, Augusta, GA, USA INDUMATHI MANOHARAN • Department of Medicine and Cancer Center, Georgia Regents University, Augusta, GA, USA Contributors
- 19. xiii PARAS KUMAR MISHRA, PH.D. • Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, USA; Department of Anesthesiology, University of Nebraska Medical Center, Omaha, NE, USA LLUÍS MONTOLIU • National Centre for Biotechnology (CNB-CSIC), Madrid, Spain; CIBERER-ISCIII, Madrid, Spain PEDRO N. MOREIRA • Mouse Biology Unit, EMBL Monterotondo, Monterotondo, Italy SHYAM SUNDAR NANDI • Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE, USA JOHNATHAN NIEVES • Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA KEVIN OAKLEY • Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, MD, USA MASATO OHTSUKA • Division of Basic Medical Science and Molecular Medicine, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan DEEPAK P. PATIL • Department of Pharmacology, Weill Medical College, Cornell University, New York, NY, USA CLAUDIA PIOVAN • Department of Molecular Virology, Immunology, and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA GANESAN RAMESH, PH.D. • Department of Medicine and Vascular Biology Center, Georgia Health Sciences University, Augusta, GA, USA PUNITHAVATHI RANGANATHAN • Department of Medicine and Vascular Biology Center, Georgia Health Sciences University, Augusta, GA, USA AMANDA RIXON • Department of Surgery, O’Brien Institute, St. Vincent’s Hospital, University of Melbourne, Melbourne, VIC, Australia; Experimental Medical and Surgical Unit (EMSU), St Vincent’s Hospital, Melbourne, VIC, Australia ANTON J.M. ROEBROEK • Laboratory for Experimental Mouse Genetics, Center for Human Genetics, KU Leuven, Leuven, Belgium KAZUHITO SAKAMOTO • Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA SARANG N. SATOOR • NHMRC Clinical Trials Centre, The University of Sydney, Camperdown, NSW, Australia LALIT SEHGAL • Advanced Centre for Treatment Research Education and Cancer, Tata Memorial Centre, Kharghar Node, Navi Mumbai, India; Department of Lymphoma/Myeloma, UT MD Anderson Cancer Center, Houston, TX, USA NAOKO SHIMA, PH.D. • Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA YOGESH SHOUCHE • Microbial Collection Centre, National Centre for Cell Science, Pune, India OLGA SIMMONS • HB Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA SHREE RAM SINGH • Stem Cell Regulation and Animal Aging Section, Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD, USA V. SIVAKAMASUNDARI • Stem Cell and Developmental Biology, Genome Institute, Singapore, Singapore Contributors
- 20. xiv MARTIN THUNEMANN • Interfakultäres Institut für Biochemie, Universität Tübingen, Tübingen, Germany ABUL USMANI • National Institute of Immunology, New Delhi, India ANDREI M. VACARU • Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA PAUL J. VERMA • Stem Cells and Reprogramming Group, Biological Engineering Laboratories, Faculty of Engineering, Monash University, Clayton, VIC, Australia; Turretfield Research Centre, South Australian Research & Development Institute, Rosedale, SA, Australia JOSEPH VITALE • Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA KAY-UWE WAGNER • Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA; Department of Genetics, Cell Biology, & Anatomy, University of Nebraska Medical Center, Omaha, NE, USA JIAN WANG • HB Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA MICHAEL D. WILLIAMS • NHMRC Clinical Trials Centre, The University of Sydney, Camperdown, NSW, Australia; Department of Surgery, O’Brien Institute, St. Vincent’s Hospital, University of Melbourne, Melbourne, VIC, Australia WILSON WONG • NHMRC Clinical Trials Centre, The University of Sydney, Camperdown, NSW, Australia XIAOHUI WU • State Key Laboratory of Genetic Engineering and Institute of Developmental Biology and Molecular Medicine, Fudan-Yale Biomedical Research Center, School of Life Sciences, Fudan University, Shanghai, China XING XING • Stem Cell and Developmental Biology, Genome Institute, Singapore, Singapore TIAN XU • State Key Laboratory of Genetic Engineering and Institute of Developmental Biology and Molecular Medicine, Fudan-Yale Biomedical Research Center, School of Life Sciences, Fudan University, Shanghai, China; Department of Genetics, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA HUI ZONG • Department of Microbiology, Immunology, and Cancer Biology, Center for Cell Signaling, University of Virginia, Charlottesville, VA, USA Contributors
- 21. Part I Mouse Genetics Techniques
- 23. 3 Shree Ram Singh and Vincenzo Coppola (eds.), Mouse Genetics: Methods and Protocols, Methods in Molecular Biology, vol. 1194, DOI 10.1007/978-1-4939-1215-5_1, © Springer Science+Business Media New York 2014 Chapter 1 Development of Pronuclear Injection-Based Targeted Transgenesis in Mice Through Cre–loxP Site-Specific Recombination Masato Ohtsuka Abstract Microinjection of DNA into the pronuclei of zygotes is the simplest and most widely used method for generating transgenic (Tg) mice. However, it is always associated with random integration of multiple copies of the transgene, resulting in unstable, low, or no transgene expression due to positional effects and/or repeat-induced gene silencing. In addition, random integration sometimes disrupts an endoge- nous gene that can affect the phenotypes of Tg mice. Our recently developed pronuclear injection-based targeted transgenesis (PITT) method enables the integration of a single-copy transgene into a predeter- mined genomic locus through Cre–loxP site-specific recombination. The PITT method enables stable and reliable transgene expression in Tg mice and is also applicable for generating knockdown mice. Therefore, the PITT method could represent next-generation transgenesis that overcomes the pitfalls of conventional transgenesis. Key words Transgenic mouse, PITT, Pronuclear injection, Targeted transgenesis, Transgene expression, Cre–loxP, Rosa26, Knockdown mouse 1 Introduction Transgenic (Tg) mice have been widely used for analyzing gene function and for creating animal models of human disease. The most commonly used method to generate Tg mice is microinjec- tion of a DNA solution into the pronuclei of fertilized eggs [pronu- clear injection (PI)]. Although this is the simplest method and has been utilized for more than three decades since Gordon et al. [1] first generated Tg mice, the PI method has some limitations such as an inability to control the transgene integration site and copy num- ber. These drawbacks often lead to unexpected and unstable trans- gene expression in Tg mice, probably due to positional effects and/or the repeat-induced gene silencing [2–4]. In addition, random transgene integration sometimes results in disruption of an endogenous gene, which may cause unexpected phenotypes.
- 24. 4 Therefore, production and analysis of multiple Tg mouse lines are always required for obtaining reliable results. Such a task is labori- ous, costly, and time consuming. Although embryonic stem (ES) cell-mediated targeted integration of transgenes can overcome these drawbacks, it is more costly and time consuming than PI-based transgenesis. Recently, we developed a novel targeted transgenesis method that enables the targeted integration of a single-copy transgene into a predetermined genomic locus through simple PI [5]. We have termed this technology as PI-based targeted transgenesis (PITT). This method is based on Cre–loxP site-specific recombina- tion. Cre (causes recombination) is an enzyme derived from bacte- riophage P1 that binds to a 34 bp region of loxP [locus of crossing over (x), P1] and catalyzes recombination between two loxP sequences. The loxP sequence consists of an 8-bp spacer region, which determines direction, between two perfect 13-bp inverted repeats. The Cre–loxP system has been widely used as a tool for modifying the genome of many model organisms, including mice. One of the most frequently utilized applications of the Cre–loxP system is the excision of the floxed DNA fragment from the genome of conditional knockout mice. In contrast, the PITT method utilizes the Cre–loxP system for insertion of the DNA of interest (DOI) into the genome (Fig. 1). For this purpose, we use the mutant loxP sequences JT15 [left element (LE)] and JTZ17 [right element (RE)] in the inverted-repeat region [6] because LE/RE double-mutated loxP, produced as a result of insertion through JT15/JTZ17 recombination, is not recognized by the Cre enzyme with high affinity, promoting the integration reaction over the excision reaction. We also used the spacer-region mutant lox2272 [7] in addition to JT15 and JTZ17 for efficient targeted integration [8]. Almost all previous reports on Cre–loxP-based Tg mice pro- duction have employed the Cre–loxP recombination reaction in ES cells [9]. In this study, we aimed to develop a targeted transgenesis method through Cre–loxP recombination in zygotes rather than ES cells. To accomplish this, we first established seed mice lines containing JT15 and lox2272 at the Rosa26 or the H2-Tw3 locus by ES cell-based gene targeting [5]. These seed mice are main- tained as homozygotes and their fertilized eggs are used for injec- tion to generate targeted Tg mice. The donor vector containing JTZ17 and lox2272 in addition to the DOI is co-injected into the eggs along with a Cre expression plasmid or iCre (codon-improved Cre recombinase) mRNA [10]. As a result, Cre–loxP recombina- tion in zygotes may integrate the DOI into the tagged Rosa26 or H2-Tw3 locus (Fig. 1). We applied the PITT method for the gen- eration of a number of gene-overexpressing Tg mice such as vari- ous fluorescent Tg mice [11]. In addition, we report the successful knockdown of the tyrosinase gene using the PITT method [5]. Masato Ohtsuka
- 25. 5 Our previous data confirmed that the expression of transgene tends to be highly reproducible and stable [11, 5]; therefore, only one germline-transmissible line is sufficient for maintenance and analy- sis. This is advantageous from the viewpoint of animal welfare because it reduces the number of mice required. Therefore, PITT could become widely used as a next-generation transgenesis method in the near future. 2 Materials 1. Donor vector: The vector contains mutant loxP sequences, such as JTZ17 and lox2272, in addition to the FRT sequence, which is used for removal of extra sequences (Figs. 1 and 2). 2.1 Vector Construction and DNA/ mRNA Preparation Fig. 1 Schematic diagram of pronuclear injection-based targeted transgenesis (PITT). The donor vector is co- injected with Cre expression plasmid or iCre mRNA into the pronuclei of fertilized eggs obtained from seed mice. In case of injection with iCre mRNA, cytoplasmic injection and pronuclear injection are performed. As a result of site-specific recombination between mutant loxPs after catalysis by the Cre enzyme, one copy of the donor vector is integrated into the predetermined genomic locus (e.g., Rosa26 or H2-Tw3) and DOIex mouse is generated. Extra sequence containing vector sequence and selection marker (neo) is removed at the FLP–FRT recombination step, resulting in the generation of the DOIΔex Targeted Transgenesis Through Pronuclear Injection
- 26. Discovering Diverse Content Through Random Scribd Documents
- 27. back
- 28. back
- 29. back
- 31. back
- 32. back
- 34. back
- 36. back
- 37. back
- 38. back
- 39. back
- 41. back
- 42. back
- 43. back
- 44. back
- 46. back
- 48. back
- 49. back
- 51. back
- 52. Welcome to our website – the ideal destination for book lovers and knowledge seekers. With a mission to inspire endlessly, we offer a vast collection of books, ranging from classic literary works to specialized publications, self-development books, and children's literature. Each book is a new journey of discovery, expanding knowledge and enriching the soul of the reade Our website is not just a platform for buying books, but a bridge connecting readers to the timeless values of culture and wisdom. With an elegant, user-friendly interface and an intelligent search system, we are committed to providing a quick and convenient shopping experience. Additionally, our special promotions and home delivery services ensure that you save time and fully enjoy the joy of reading. Let us accompany you on the journey of exploring knowledge and personal growth! ebookfinal.com