![mystic creed, and he is said to be one of the few
students of him who have ever read through all the
Latin writings of the Swedish theologian. He soon
resigned his curacy, and for a short time he preached
in the Swedenborgian temple in Peter Street. There
he seems not to have found the freedom of opinion
and breadth in teaching he had expected, and he
determined to propagate his own convictions,
independently of other authority. In the year 1800 he
built, at his own expense, Christ Church, in King
Street, Salford—the first meeting-place of the
reformed church.[261]
His extraordinary eloquence and
ability, as well as earnestness of purpose, quickly
attracted a large audience, and may well have
brought to recollection the style and matter of the
great orator of Constantinople of the fourth century.
One characteristic of his Church—perhaps unique at
that time—was the non-appropriation of sittings.
Another unfashionable opinion held by him was the
Pauline one of the obligation upon Christian
preachers to maintain themselves by some “secular”
labour, and he therefore kept a boarding school,
which attained extensive proportions. In this college
some zealous and able men, who afterwards were
ordained by him to carry on a truly beneficent
ministry, assisted in the work of teaching, of whom
the names of Metcalfe, Clark, and Schofield are](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-28-320.jpg)
![collected the most various matter illustrative of
passages in the Bible, and in defence of his own
interpretation of them, was there printed. It is, as his
biographer has well described it, “a lasting memorial
of his wide reading and research—travellers, lawyers,
poets, physicians, all are pressed into his service—
the whole work forming a large quarto common-
place book filled with reading as delightful as it is
discursive. Some of his minor writings have also been
printed. He was, besides his theological erudition, a
practical chemist and astronomer, and he caused the
dome of the church in King Street to be fitted up for
the joint purposes of an observatory and a
laboratory. His microscope is still preserved in the
Peel Park Museum. His valuable library, which at one
time was accessible to the public on easy terms, is
now deposited in the new Bible Christian Church in
Cross Lane. The books collected exhibit the strong
mind which brought them together for its own uses.
This library is the workshop in which he wrought out
a new mode of life and a new theory of doctrine—
with these instruments he moulded minds like that of
Brotherton, and so his influence has worked in many
unseen channels.” He died in 1816, and is buried in
front of his chapel, in King Street, Salford.[262]](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-30-320.jpg)
![other dietary reformers, the young converts
encountered much opposition from their family and
friends, who attempted at one moment ridicule, at
another dissuasion, by appealing to medical
authority. Unmoved from their purpose, they
continued unshaken in their convictions.
“They assured me,” he writes at a later period, “that I was
rapidly sinking into a consumption, and tried various other
methods to induce me to return to the customary dietetic
habits of society; but their efforts proved ineffectual. Some
predicted my death in three or four months; and others, on
hearing me attempt to defend my course, hesitated not to tell
me I was certainly suffering from mental derangement, and,
if I continued to live without flesh-food much longer, would
unquestionably have to be shut up in some insane asylum. All
was unavailing. Instead of sinking into consumption, I gained
several pounds in weight during the first few weeks of my
experiment. Instead of three or four months bringing me to
the silent grave, they brought me to the matrimonial altar.
“She [his wife] fully coincided with me in my views on
vegetable diet, and, indeed, on all other important points was
always ready to defend them to the best of her ability—
studied to show our acquaintances, whenever they paid us a
visit, that we could live, in every rational enjoyment, without
the use of flesh for food. As she was an excellent cook, we
were never at a loss as to what we should eat. We
commenced housekeeping in January, 1810, and, from that
date to the present time, we have never had a pound of
flesh-meat in our dwelling, have never patronised either
slaughter-houses or spirit shops.
“When, again, in the course of time we were about to be
blessed with an addition to our family, a renewed effort was](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-32-320.jpg)
![made. We were assured it was impossible for my wife to get
through her confinement without some more strengthening
food. Friends and physicians were alike decided upon that
point. We were, notwithstanding, unmoved and faithful to our
principles. Next we were told by our kind advisers that the
little stranger could not be sufficiently nourished unless the
mother could eat a little ‘meat’ once a day; or, if not that,
drink a pint or half a pint of ale daily. To both proposals my
wife turned a deaf ear; and both she and the child did
exceedingly well.[263] It may be proper to add here [remarks
the biographer], that the ‘little stranger’ above referred to is
the author of this Memoir,—that he is in the fifty-sixth year of
his age, that he has never so much as tasted animal food, nor
used intoxicating drinks of any kind, and that he is hale and
hearty.”
These experiences, it is scarcely necessary to
remark, in the lives of followers of reformed dietetics,
have been not seldom repeated.
In the Academy of Sciences, instituted by Dr.
Cowherd, Metcalfe was invited to assume the
direction of the “classical” department (1811). In the
same year he took “Orders,” and, at the solicitation
of the secessionists from the Swedenborgian
Communion (which, with some inconsistency, seems
to have looked with indifference, or even dislike,
upon the principles of akreophagy), he officiated at
Adingham, in Yorkshire. By the voluntary aid of one
of his admirers a church was built, to which was
added a commodious school-room. He then resigned](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-33-320.jpg)
![in his life-work; and, as the first who systematically
taught the truths of reformed dietetics in the “New
World,” he has deserved the unceasing gratitude of
all sincere reformers in the United States, and,
indeed, throughout the globe. By all who knew him
personally he was as much loved as he was
esteemed, and the newspapers of the day bore
witness to the general lamentation for his loss.[264]](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-40-320.jpg)
![learn the trade of paper-making. “He is described as
handsome, clever, and imaginative. ‘I had heard,’ he
says, ‘of noble deeds, and longed to follow in the
field of fame.’ Ill health soon obliged his return to the
country, and at sixteen symptoms of consumption
appeared. Various occupations were tried until the
time, when about twenty years of age, he
commenced as a teacher of youth, proving highly
successful with his pupils. Again ill-health obliged the
abandonment of this pursuit.”[265]
At the age of thirty-two he married, and soon
after became a preacher in the Presbyterian Church.
Deeply interested in the question of “Temperance,”
he was invited to lecture for that cause by the
Pennsylvania Society (1830). He now began the
study of physiology and comparative anatomy, in
which his interest was unremitting. These important
sciences were used to good effect in his future
dietetic crusade. At this time he came in contact with
Metcalfe, by whom he was confirmed in, if not in the
first instance converted to, the principles of radical
dietary reform. “He was soon led to believe that no
permanent cure for intemperance could be found,
except in such change of personal and social
customs as would relieve the human being from all
desire for stimulants. This idea he soon applied to
medicine, so that the prevention and cure of disease,](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-42-320.jpg)
![as well as the remedy for intemperance, were seen
to consist mainly in the adoption of correct habits of
living, and the judicious adaptation of hygienic
agencies. These ideas were elaborated in an Essay
on the Cholera (1832), and a course of lectures
which were delivered in various parts of the country,
and subsequently published under the title of
Lectures on the Science of Human Life (2 vols.,
Boston, 1839). This has been the leading text-book
of all the dietetic and nearly all the health reformers
since.”[266]
The Science of Human Life is one of the most
comprehensive as well as minute text books on
scientific dietetics ever put forth. If it errs at all, it
errs on the side of redundancy—a feature which it
owes to the fact that it was published to the world as
it was orally given. It therefore well bears
condensation, and this has been judiciously done by
Mr. Baker, whose useful edition is probably in the
hands of most of our readers. Graham was also the
author of a treatise on Bread and Bread-Making, and
“Graham bread” is now universally known as one of
the most wholesome kinds of the “staff of life.”
Besides these more practical writings, for some time
before his death he occupied his leisure in the
production of a Philosophy of Sacred History, the
characteristic idea of which seems to have been to](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-43-320.jpg)
![of courage and enterprise, it is concluded that vegetable food
is the cause. Yet examination might have shown that causes
fully adequate to these effects existed, which not only
exonerated the diet, but made it appear that the vegetable
diet had a redeeming effect, and was the means by which the
nation was saved from a worse condition.
“The fact that individuals have attained a great age in
certain habits of living is no evidence that those habits are
favourable to longevity. The only use which we can make of
cases of extraordinary old age, is to show how the human
constitution is capable of sustaining the vital economy, and
resisting the causes which induce death.
“If we ask how we must live to secure the best health and
longest life, the answer must be drawn from physiological
knowledge; but if we ask how long the best mode of living
will preserve life, the reply is, Physiology cannot teach you
that. Probably each aged individual has a mixture of good and
bad habits, and has lived in a mixture of favourable and
unfavourable circumstances. Notwithstanding apparent
diversity, there is a pretty equal amount of what is salutary in
the habits and circumstances of each. Some have been
‘correct’ in one thing, some in another. All that is proved by
instances of longevity in connexion with bad habits is, that
such individuals are able to resist causes that have, in the
same time, sent thousands of their fellow-beings to an
untimely grave; and, under a proper regimen, they would
have sustained life, perhaps, a hundred and fifty years.
“Some have more constitutional [or inherited] powers to
resist the causes of disease than others, and, therefore, what
will destroy the life of one may be borne by another a long
time without any manifestations of immediate injury. There
are, also, constitutional peculiarities, but these are far more
rare than is generally supposed. Indeed, such may, in almost
every case, be overcome by a correct regimen. So far as the](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-47-320.jpg)
![general laws of life and the application of general principles of
regimen are considered, the human constitution is one: there
are no constitutional differences which will not yield to a
correct regimen, and thus improve the individual.
Consequently, what is best for one is best for all.... Some are
born without any tendency to disease while others have the
predisposition to particular diseases of some kind. But
differences result from causes which man has the power to
control, and it is certain that all can be removed by
conformity to the laws of life for generations, and that the
human species can be brought to as great uniformity, as to
health and life, as the lower animals.”
With Hufeland, Flourens, and other scientific
authorities, he maintains that:—
“Physiological science affords no evidence that the human
constitution is not capable of gradually returning to the
primitive longevity of the species. The highest interests of our
nature require that youthfulness should be prolonged. And it
is as capable of being preserved as life itself, both depending
on the same conditions. If there ever was a state of the
human constitution which enabled it to sustain life [much
beyond the present period], that state involved a harmony of
relative conditions. The vital processes were less rapid and
more complete than at present, development was slower,
organisation more perfect, childhood protracted, and the
change from youth to manhood took place at a greater
remove from birth. Hence, if we now aim at long life, we can
secure our object only by conformity to those laws by which
youthfulness is prolonged.”
As for the omnivorousness of the human animal:
—](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-48-320.jpg)
![The ourang-outang, on being domesticated, readily learns
to eat animal food. But if this proves that animal to be
omnivorous, then the Horse, Cow, Sheep, and others are all
omnivorous, for everyone of them is easily trained to eat
animal food. Horses have frequently been trained to eat
animal food,[267] and Sheep have been so accustomed to it as
to refuse grass. All carnivorous animals can be trained to a
vegetable diet, and brought to subsist upon it, with less
inconvenience and deterioration than herbivorous or
frugivorous animals can be brought to live on animal food.
Comparative anatomy, therefore, proves that Man is naturally
a frugivorous animal, formed to subsist upon fruits, seeds,
and farinaceous vegetables.[268]
The stimulating, or alcoholic, property of flesh
produces the delusion that it is, therefore, the most
nourishing:—
“Yet by so much as the stimulation exceeds that which is
necessary for the performance of the functions of the organs,
the more does the expenditure of vital powers exceed the
renovating economy; and the exhaustion which succeeds is
commensurate with the excess. Hence, though food which
contains the greatest proportion of stimulating power causes
a feeling of the greatest strength, it also produces the
greatest exhaustion, which is commensurately importunate
for relief; and, as the same food affords such by supplying
the requisite stimulation, their feelings lead the consumers to
believe that it is most strengthening.... Those substances, the
stimulating power of which is barely sufficient to excite the
digestive organs in the appropriation of nourishment, are
most conducive to vital welfare, causing all the processes to
be most perfectly performed, without any unnecessary
expenditure, thus contributing to health and longevity.](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-49-320.jpg)
![“Flesh-meats average about thirty-five per cent of
nutritious matter, while rice, wheat, and several kinds of pulse
(such as lentils, peas, and beans), afford from eighty to
ninety-five per cent; potatoes afford twenty-five per cent of
nutritious matter. So that one pound of rice contains more
nutritious matter than two pounds and a half of flesh meat;
three pounds of whole meal bread contain more than six
pounds of flesh, and three pounds of potatoes more than two
pounds of flesh.”
That the human species, taken in its entirety, is
no more carnivorous de facto than it could be de
jure, is apparent on the plain evidence of facts. In all
countries of our Globe, with the exception of the
most barbarous tribes, it is, in reality, only the ruling
and rich classes who are kreophagist. The Poor have,
almost everywhere, but the barest sufficiency even
of vegetable foods:—
“The peasantry of Norway, Sweden, Denmark, Germany,
Turkey, Greece, Italy, Switzerland, France, Spain, England,
Scotland, Ireland, a considerable portion of Prussia, and other
parts of Europe subsist mainly on non-flesh foods. The
peasantry of modern Greece [like those of the days of
Perikles] subsist on coarse brown bread and fruits. The
peasantry in many parts of Russia live on very coarse bread,
with garlic and other vegetables, and, like the same class in
Greece, Italy, &c., they are obliged to be extremely frugal
even in this kind of food. Yet they are [for the most part]
healthy, vigorous, and active. Many of the inhabitants of
Germany live mainly on rye and barley, in the form of coarse
bread. The potato is the principal food of the Irish peasantry,
and few portions of the human family are more healthy,](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-50-320.jpg)
![athletic, and active, when uncorrupted by intoxicating
substances [and, it may be added, when under favourable
political and social conditions]. But alcohol, opium, &c.
[equally with bad laws] have extended their blighting
influence over the greater portion of the world, and nowhere
do these scourges so cruelly afflict the self-devoted race as in
the cottages of the poor, and when, by these evils and
neglect of sanitation, &c., diseases are generated, sometimes
epidemics, we are told that these things arise from their poor,
meagre, low, vegetable diet. Wherever the various sorts of
intoxicating substances are absent, and a decent degree of
cleanliness is observed, the vegetable diet is not thus
calumniated.
“That portion of the peasantry of England and Scotland
who subsist on their barley and oatmeal bread, porridge,
potatoes, and other vegetables, with temperate, cleanly
habits [and surroundings], are able to endure more fatigue
and exposure than any other class of people in the same
countries. Three-fourths of the whole human family, in all
periods of time [excepting, perhaps, in the primitive wholly
predatory ages] have subsisted on non-flesh foods, and when
their supplies have been abundant, and their habits in other
respects correct, they have been well nourished.”
That the sanguinary diet and savagery go hand in
hand, and that in proportion to the degree of
carnivorousness is the barbarous or militant
character of the people, all History, past and present,
too clearly testifies. Nor are the carnivorous tribes
conspicuous by their cruel habits only:—
“Taking all flesh-eating nations together, though some,
whose other habits are favourable, are, comparatively, well-](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-51-320.jpg)
![formed, as a general average they are small, ill-formed races;
and taking all vegetable-eating nations, though many, from
excessive use of narcotics, and from other unfavourable
circumstances, are comparatively small and ill-formed, as a
general average they are much better formed races than the
flesh-eaters.[269] It is only among those tribes whose habits
are temperate, and who subsist on the non-flesh diet, that
the more perfect specimens of symmetry are found.
“Not one human being in many thousands dies a natural
death. If a man be shot or poisoned we say he dies a violent
death, but if he is ill, attended by physicians, and dies, we say
he dies a ‘natural’ death. This is an abuse of language—the
death in the latter case being as truly violent as if he had
been shot. Whether a man takes arsenic and kills himself, or
by small doses or other means, however common, gradually
destroys life, he equally dies a violent death. He only dies a
natural death who so obeys the laws of his nature as by
neither irritation nor intensity to waste his energies, but
slowly passes through the changes of his system to old age,
and falls asleep in the exhaustion of vitality.”[269]
With Flourens he adduces a number of instances
both of individuals and of communities who have
attained to protracted ages by reason of a pure diet.
He afterwards proceeds to prove from comparative
physiology and anatomy, and, in particular, from the
conformation of the human teeth and stomach
(which, by an astounding perversion of fact, are
sometimes alleged to be formed carnivorously, in
spite of often-repeated scientific authority, as well as
of common observation), the natural frugivorous
character of the human species, and he quotes](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-52-320.jpg)
![Linné, Cuvier, Lawrence, Bell, and many others in
support of this truth.[270]](https://image.slidesharecdn.com/9654-250616174358-420fcc76/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-53-320.jpg)
Transgenesis Techniques Principles and Protocols 3rd Edition Leonie Ringrose (Auth.)
- 1. Transgenesis Techniques Principles and Protocols 3rd Edition Leonie Ringrose (Auth.) download pdf https://ebookultra.com/download/transgenesis-techniques-principles-and- protocols-3rd-edition-leonie-ringrose-auth/ Visit ebookultra.com today to download the complete set of ebook or textbook!
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- 5. Transgenesis Techniques Principles and Protocols 3rd Edition Leonie Ringrose (Auth.) Digital Instant Download Author(s): Leonie Ringrose (auth.), Elizabeth J. Cartwright (eds.) ISBN(s): 9781603270199, 1603270191 Edition: 3 File Details: PDF, 4.07 MB Year: 2009 Language: english
- 6. ME T H O D S I N MO L E C U L A R BI O L O G Y ™ Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For other titles published in this series, go to www.springer.com/series/7651
- 7. i i i Transgenesis Techniques Principles and Protocols Third Edition Edited by Elizabeth J. Cartwright CardiovascularMedicine,UniversityofManchester,Manchester,UK
- 8. iv ISSN: 1064-3745 e-ISSN: 1940-6029 ISBN: 978-1-60327-018-2 e-ISBN: 978-1-60327-019-9 DOI: 10.1007/978-1-60327-019-9 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2009929336 © Humana Press, a part of Springer Science+Business 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 Science+Business 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. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) Editor Elizabeth J. Cartwright Cardiovascular Medicine University of Manchester Manchester UK
- 9. v To Dan, Edward and William with love.
- 10. Preface One of the major challenges currently facing the scientific community is to understand the function of the 20,000–25,000 protein-coding genes that were revealed when the human genome was fully sequenced. This book details the transgenic techniques that are currently used to modify the genome in order to extend our understanding of the in vivo function of these genes. Since the advent of transgenic technologies, the mouse has become by far the most popular model in which to study mammalian gene function. This is due to not only its genetic similarity to humans but also its physiological and, to a certain extent, its anatomical similarities. Whilst a large proportion of this book is dedicated to the use of the mouse in transgenesis, the mouse is certainly not the only model to provide essential information regarding gene function. A number of other valuable models are used in transgenic studies including Drosophila, C. elegans, Xenopus, zebrafish, and rat. For each of these species, a chapter in this book is dedicated to highlighting how each is particularly suited, for example, to the study of embryonic development, physiological function of genes and to study orthologs of human disease genes. These chapters give detailed practical descriptions of animal production, construct design, and gene transfer techniques; recently developed methods will be described along with highly established classical techniques. A number of chapters in this book are dedicated to the generation of genetically modified mice by the present classic techniques of injection of exogenous DNA into the pronuclei of fertilised eggs and by gene targeting using homologous recombination in embryonic stem cells. These chapters, as with all the others in the book, have been specifically written for this edition of Transgenesis and so contain up-to-date details of the practices in the field. Chapters are included describing optimal transgene and construct design, in-depth technical details for pronuclear microinjection of transgenes and associated surgical techniques, details for the optimal conditions in which to culture embryonic stem cells in order to maintain their pluripotent state, and methods for targeting these cells. A combination of chapters (Chaps. 13–15) describe how to generate chimaeras by microinjection of targeted ES cells into blastocysts or by morula aggregation, and the surgical techniques required to transfer the resulting embryos. For a number of years, the use of Cre/loxP and flp/frt recombination systems has gained in popularity; Chap. 16 describes their use and introduces other state-of-the-art site- specific recombination systems that can be used to manipulate the mouse genome. The generation and use of Cre-expressing transgenic lines are described in Chap. 17. One chapter of the book highlights the large-scale international efforts that are being made to systematically knockout every gene in the genome. The remaining chapters detail the breeding and husbandry skills required to successfully propagate a transgenic line and the increasingly essential methods for cryopreserving a mouse line and recovering lines from frozen stocks. This book is a comprehensive practical guide to the generation of transgenic animals and is packed full of handy hints and tips from the experts who use these techniques on a vii
- 11. viii Preface day-to-day basis. It is designed to become an invaluable source of information in any lab currently involved in transgenic techniques, as well as for researchers who are newcomers to the field. This book also provides essential background information for scientists who work with these models but have not been involved in their generation. On a personal note, it has been a great pleasure to edit this latest edition of Transgenesis. Firstly, I learnt many of my skills from reading earlier editions of the book and I hope that this edition will help and inspire many others. Secondly, I have been privileged to work with the exceptionally talented researchers in the transgenesis field who have contributed to this book. Manchester, UK Elizabeth J. Cartwright
- 12. ix Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi PART I TRANSGENESIS IN VARIOUS MODEL SYSTEMS 1. Transgenesis in Drosophila melanogaster. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Leonie Ringrose 2. Transgenesis in Caenorhabditis elegans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Matthias Rieckher, Nikos Kourtis, Angela Pasparaki, and Nektarios Tavernarakis 3. Transgenesis in Zebrafish with the Tol2 Transposon System . . . . . . . . . . . . . . . . . 41 Maximiliano L. Suster, Hiroshi Kikuta, Akihiro Urasaki, Kazuhide Asakawa, and Koichi Kawakami 4. Generation of Transgenic Frogs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Jana Loeber, Fong Cheng Pan, and Tomas Pieler 5. Pronuclear DNA Injection for the Production of Transgenic Rats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Jean Cozzi, Ignacio Anegon, Valérie Braun, Anne-Catherine Gross, Christel Merrouche, and Yacine Cherifi PART II TRANSGENESIS IN THE MOUSE 6. Cell-Type-Specific Transgenesis in the Mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 James Gulick and Jeffrey Robbins 7. Transgene Design and Delivery into the Mouse Genome: Keys to Success . . . . . . 105 Lydia Teboul 8. Overexpression Transgenesis in Mouse: Pronuclear Injection . . . . . . . . . . . . . . . . 111 Wendy J.K. Gardiner and Lydia Teboul 9. Gene-Targeting Vectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 J. Simon C. Arthur and Victoria A. McGuire 10. Gene Trap: Knockout on the Fast Lane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Melanie Ullrich and Kai Schuh 11. Culture of Murine Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Ivana Barbaric and T. Neil Dear 12. Targeting Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Roland H. Friedel 13. Generation of Chimeras by Microinjection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Anne Plück and Christian Klasen
- 13. x Contents 14. Generation of Chimeras by Morula Aggregation . . . . . . . . . . . . . . . . . . . . . . . . . 219 Anne Plück and Christian Klasen 15. Surgical Techniques for the Generation of Mutant Mice . . . . . . . . . . . . . . . . . . . 231 Anne Plück and Christian Klasen 16. Site-Specific Recombinases for Manipulation of the Mouse Genome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Marie-Christine Birling, Françoise Gofflot, and Xavier Warot 17. Cre Transgenic Mouse Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Xin Wang 18. Large-Scale Mouse Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 Elizabeth J. Cartwright 19. Dedicated Mouse Production and Husbandry . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 Lucie Vizor and Sara Wells 20. Biological Methods for Archiving and Maintaining Mutant Laboratory Mice. Part I: Conserving Mutant Strains . . . . . . . . . . . . . . . . 301 Martin D. Fray 21. Biological Methods for Archiving and Maintaining Mutant Laboratory Mice. Part II: Recovery and Distribution of Conserved Mutant Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 Martin D. Fray Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
- 14. Contributors IGNACIO ANEGON • INSERM – Institut National de la Santé et de la Recherche Médicale, Nantes, France J. SIMON C. ARTHUR • MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, UK KAZUHIDE ASAKAWA • Division of Molecular and Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan IVANA BARBARIC • Department of Biomedical Science, University of Sheffield, Sheffield, UK MARIE-CHRISTINE BIRLING • Institut Clinique de la Souris – Mouse Clinical Institute (ICS-MCI), Illkirch, France VALÉRIE BRAUN • genOway SA, Lyon, France ELIZABETH J. CARTWRIGHT • Cardiovascular Medicine, University of Manchester, Manchester, UK YACINE CHERIFI • genOway SA, Lyon, France JEAN COZZI • genOway SA, Lyon, France T. NEIL DEAR • Leeds Institute of Molecular Medicine, St. James’s University Hospital, Leeds, UK MARTIN FRAY • Frozen Embryo & Sperm Archive (FESA), Medical Research Council, Mammalian Genetics Unit, Harwell, UK ROLAND H. FRIEDEL • Institute of Developmental Genetics, Helmholtz Center Munich, Neuherberg, Germany WENDY J.K. GARDINER • Mary Lyon Centre, Medical Research Council, Harwell, UK FRANÇOISE GOFFLOT • Institut Clinique de la Souris – Mouse Clinical Institute (ICS-MCI), Illkirch, France ANNE-CATHERINE GROSS • genOway SA, Lyon, France JAMES GULICK • Molecular Cardiovascular Biology, Cincinnati Children’s Hospital, University of Cincinnati, Cincinnati, OH, USA KOICHI KAWAKAMI • Division of Molecular and Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan HIROSHI KIKUTA • Division of Molecular and Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan CHRISTIAN KLASEN • Transgenic Service, European Molecular Biology Laboratory, Heidelberg, Germany NIKOS KOURTIS • Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece JANA LOEBER • Department of Developmental Biochemistry, University of Goettingen, Goettingen, Germany xi
- 15. VICTORIA A. MCGUIRE • MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, UK CHRISTEL MERROUCHE • genOway SA, Lyon, France FONG CHENG PAN • Vanderbilt University Program in Developmental Biology and Department of Cell and Biology, Vanderbilt University Medical Center, Nashville, TN, USA ANGELA PASPARAKI • Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece TOMAS PIELER • Department of Developmental Biochemistry, University of Goettingen, Goettingen, Germany ANNE PLÜCK • Centre for Mouse Genetics, Institute for Genetics, University of Cologne, Cologne, Germany MATTHIAS RIECKHER • Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece LEONIE RINGROSE • IMBA – Institute of Molecular Biotechnology GmbH, Vienna, Austria JEFFREY ROBBINS • Molecular Cardiovascular Biology, Cincinnati Children’s Hospital, University of Cincinnati, Cincinnati, OH, USA KAI SCHUH • Institute of Physiology I, University of Wuerzburg, Wuerzburg, Germany MAXIMILIANO L. SUSTER • Division of Molecular and Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan NEKTARIOS TAVERNARAKIS • Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece LYDIA TEBOUL • Mary Lyon Centre, Medical Research Council, Harwell, UK MELANIE ULLRICH • Institute of Physiology I, University of Wuerzburg, Wuerzburg, Germany AKIHIRO URASAKI • Division of Molecular and Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan LUCIE VIZOR • Medical Research Council, Harwell, UK XIN WANG • Faculty of Life Sciences, University of Manchester, Manchester, UK XAVIER WAROT • EPFL FSV – École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland SARA WELLS • Medical Research Council, Harwell, UK xii xii Contributors
- 16. Part I Transgenesis in Various Model Systems
- 17. Chapter 1 Transgenesis in Drosophila melanogaster Leonie Ringrose Summary Transgenesis in Drosophila melanogaster relies upon direct microinjection of embryos and subsequent crossing of surviving adults. The necessity of crossing single flies to screen for transgenic events limits the range of useful transgenesis techniques to those that have a very high frequency of integration, so that about 1 in 10 to 1 in 100 surviving adult flies carry a transgene. Until recently, only random P-element transgenesis fulfilled these criteria. However, recent advances have brought homologous recombination and site-directed integration up to and beyond this level of efficiency. For all transgenesis techniques in Drosophila melanogaster, microinjection of embryos is the central procedure. This chapter gives a detailed protocol for microinjection, and aims to enable the reader to use it for both site-directed inte- gration and for P-element transgenesis. Key words: Drosophila melanogaster, Embryo, Microinjection, Transgenic, Recombination, Inte- gration, Homologous recombination, phiC31/integrase, Site-directed integration, P-element Transgenesis in Drosophila melanogaster has undergone something of a revolution in the last few years. The classical technique of random P-element-mediated transgenesis has recently been sup- plemented by two novel technologies: homologous recombi- nation and ΦC31 integration (for reviews, see (1) and (2)). In P-element transgenesis (3), a modified transposon vector is used in combination with transient expression of the P transposase enzyme to generate several fly lines with different insertion sites in the genome. These insertions are subsequently mapped and characterised. P-element insertions have been invaluable for mutagenesis screens, but until recently, this was also the only 1. Introduction Elizabeth J. Cartwright (ed.), Transgenesis Techniques, Methods in Molecular Biology, vol. 561 DOI 10.1007/978-1-60327-019-9_1, © Humana Press, a part of Springer Science+Business Media, LLC 2009 3
- 18. 4 Ringrose method available for introducing a transgene of choice into the Drosophila genome. The random nature of P-element insertions has several drawbacks for transgene analysis. Mapping of inser- tion sites is time consuming, and transgene expression levels are subject to genomic position effects, making it difficult to draw comparisons between different constructs. A recently developed alternative to random insertion is homologous recombination (4, 5). This involves inserting a donor construct at random into the genome by P-element trans- genesis, and in subsequent generations, mobilising the donor construct to the correct locus by homologous recombination. This technique had long been lacking to Drosophilists, but has not replaced P-element transgenesis as the method of choice for routine transgene analysis, because both the cloning of donor constructs and the generation of homologous recombinants are more time consuming than for P-element transgenesis. Recently, ΦC31 integration has been developed (6). This technique allows rapid and efficient generation of site-specific integrants, and relies upon ‘docking site’ fly lines, which carry a single recognition site (attP) for the phage ΦC31 integrase enzyme, previously introduced into the genome by P-element transgenesis. A donor plasmid carrying a second recognition site (attB) and a source of integrase enzyme is used to generate flies in which the donor plasmid docks to the genomic site. Integration events are highly specific, as the attP site is 39 bp long and does not occur at random in the Drosophila genome. Many mapped and characterised docking site lines are now available (see Note 1), and ΦC31 integration is rapidly becoming widely used for many transgenic applications. All these transgenic techniques rely upon microinjection of embryos as a first step. In early Drosophila embryogenesis, the nuclei share a common cytoplasm for the first nine divisions. Directly after the tenth division, the first cells to become sep- arated are the pole cells, which will later form the adult germ line. Transgenic animals are made by microinjecting DNA and a source of enzyme (P-transposase or ΦC31 integrase, see Note 2) into the posterior of the embryo where the pole cells will form, at an early stage before they have become separated from the common cytoplasm. DNA can enter the nuclei and is integrated into the genome of some cells. Embryos are allowed to mature and the adults are outcrossed to screen for transgenic flies in the next generation. This chapter gives a detailed description of microinjection, from preparing DNA to screening for transformants. The main protocol deals with ΦC31 integration as we perform it in our laboratory. Alternatives for both ΦC31 and P element transgen- esis are given in the notes.
- 19. Transgenesis in Drosophila melanogaster 5 1. Donor plasmid containing attB site and transgene of interest (see Note 3). 2. Helper plasmid expressing ΦC31 integrase (see Note 2). Midi- or miniprep kit for preparation of plasmid DNA (Qiagen). 3. Absolute ethanol. 4. 3 M NaOAc, pH 5.2. 5. Sterile distilled water. 1. Capillaries: borosilicate glass capillaries, 1.2 mm × 0.94 mm 2. Needle puller: P-97 micropipette puller (Sutter instruments). 3. Needle grinder: Narishige microgrinder EG-400. 1. Fly line containing genomic attP site (see Note 1). 2. Fly bottles. 3. Fresh yeast paste: cubes of fresh baker’s yeast cubes are obtain- able from large supermarkets. They can be frozen and stored at −20°C for several months. Thaw at room temperature and mix with a little water to give a thick paste. 4. Dried yeast: Mix instant yeast granules with water to give a thick paste. Both fresh and dried yeast paste can be kept at 4°C for up to a week. Do not seal the container tightly, as the paste will expand. 5. Fly cages: PVC plastic tubing of either 50 mm or 90 mm diameter is cut into 100–150 mm sections and sealed at one end with nylon or metal mesh. The other end fits onto to a 50-mm or 90-mm agar plate, which is taped in place for egg collection. 6. Agar plates: Add 18 g agar to 600 mL tap water and bring to boiling point by microwaving. Dissolve 10 g sucrose in 300 mL tap water, heating a little if necessary. Add the sucrose solution to the agar, add 3.5 mL 100% acetic acid and mix well. Pour into petri dishes (90 mm or 50 mm) and allow to cool. Store for 1 day at room temperature to dry before using. Plates can be stored wrapped in plastic at 4°C for several weeks. About 16–20 plates per day of injection are required per cage (see Note 5). 1. Filtration apparatus consisting of glass funnel, filter support, stopper, sidearm flask, and clamp, suitable for 50-mm mem- brane filters. Attach the apparatus to water tap as shown in Fig. 1. 2. Materials 2.1. Preparation of DNA 2.2. Preparation of Injection Needles (see Note 4) 2.3. Preparation of Flies for Egg Laying 2.4. Dechorionation and Dessication of Embryos
- 20. 6 Ringrose 2. Bleach solution: mix 50 mL household bleach (2.8% hypochlorite) with 50 mL sterile distilled water. Make fresh every day. Wear a lab coat and gloves when handling bleach, as it bleaches clothes upon contact and is harmful to skin. 3. Membrane filters: mixed cellulose ester membrane filters, black with white grid marking. Circular, 50-mm diameter, 0.6-μm pore size (Schleicher and Schuell, type ME 26/31 ST). 4. Binocular dissection microscope. 5. Fine stiff paintbrush with nylon hairs: cut away hairs until only a few remain, for use in aligning embryos. 6. Dissection needle. 7. Forceps. 8. Microscope slides: use slides with frosted part for labelling, such as Superfrost plus (Fisher). 9. Coverslips: 24 × 24 mm. 10. Embryo glue: Make three balls of 2.5-m Scotch tape Magic 810 (3 M). Add these to 30 mL heptane in a 50-mL falcon tube. Shake vigorously at 28°C for 24 h. Cut a hole in the bottom of the falcon tube and drain solution into a small glass bottle. This glue keeps for several months at room tem- perature (see Note 6). 11. Drying chamber: 150-mm petri dish containing orange self- indicating silica gel granules: check that the silica gel gran- ules are orange; if they are not then they are saturated and no longer effective for drying embryos. Change to fresh granules. 12. Halocarbon oil: Voltalef 10S halocarbon oil, or halocarbon 700 oil (Sigma). Fig. 1. Filtration apparatus.
- 21. Transgenesis in Drosophila melanogaster 7 1. Microscope: Either a compound or inverted microscope is suitable for injection. We use a Zeiss Axiovert 200 inverted microscope with ×10 objective and ×10 oculars. 2. Micromanipulator and needle holder (Narishige). 3. Microinjection system: Femtojet 5247 programmable microin- jector with integrated pressure supply (Eppendorf) (see Note 7). 4. Microloader pipette tips (Eppendorf). 1. Humid box: sealable plastic sandwich box containing damp paper towels. 2. 50-mm Petri dishes. 3. 18 mm × 18 mm cover slips. 4. Flies for crossing to surviving adults: w- or appropriate bal- ancer lines. 5. Fly vials. 1. Prepare donor and helper plasmids in advance. Use midi- or miniprep (Qiagen quality) DNA. Do not elute the DNA in the buffer provided, as it contains Tris buffer, which is harmful to embryos. Instead, elute in sterile distilled water (see Note 8). 2. Check the concentration of eluted DNA. If the concentration is sufficient, make an injection mix at 250 ng/μL of donor vector plus 600 ng/μL of helper, in sterile distilled water (see Note 9). 3. If the DNA concentration is too low, precipitate the DNA: Add 0.1 volume of 3 M NaOAc, pH 5.2, and two volumes of abso- lute ethanol. Incubate at –20°C overnight. Centrifuge at 4°C for 10 min at 14,000 × g. Remove the supernatant, add 70% ethanol to the pellet, and centrifuge at 4°C for 5 min at 14,000 × g. Air dry the pellet and resuspend in sterile distilled water. 4. Plasmids and injection mixes can be stored indefinitely at −20°C. For DNA stored in water, however, the absence of a buffering agent may lead to degradation upon repeated freez- ing and thawing (see Note 8). 1. Before beginning to inject, prepare a supply of needles. We use a needle puller (P-97, Sutter instruments) with the follow- ing settings: Heat = 595; Pull = 70; Vel = 80). Insert a glass capillary into the needle puller, close the lid, and press ‘pull’. This makes two needles from each capillary that are closed at the tapered end (see Note 4). 2.5. Microinjection of Embryos 2.6. Further Handling and Screening for Transgenics 3. Methods 3.1. Preparation of DNA 3.2. Preparation of Injection Needles
- 22. 8 Ringrose 2. Open the needles by grinding in the needle grinder. Insert the needle into the holder at an angle of 40° to the grind- stone. Keeping a constant flow of water over the grindstone, lower the needle onto the grindstone till the tip bends very slightly and water rises up into the needle. Immediately the water enters; stop moving the needle and allow to grind for 20 s (see Note 9). 1. Expand the fly line that is to be injected to give six bottles. Flip all six each week if large-scale injections are planned. Use flies that are 1-week old and well fed for the best egg laying. 2. One week before injection: flip adult flies every 2 days into bottles with fresh yeast paste. This feeds them optimally, so females lay a lot of eggs. Keep these bottles at 18°C. 3. Two days before injection: transfer flies to cages (use 4–6 bot- tles per 90-mm-diameter cage). Add a little dried yeast paste on a small square of paper (this facilitates later removal) onto the plates and place the cages at 25°C. Change the plates every 24 h and discard them. This acclimatises flies to the cage envi- ronment. 4. On the day of injection: Ensure that plates are at room tem- perature. Change the overnight plate, and wipe the inner rim of the cage to remove any first instar larvae. Add a very small spot of yeast paste on a square of paper to the centre of each new plate. Change the first plate after 1 h, and discard it. This is because females may keep fertilised eggs for some time before laying them. Use the subsequent plates for collections. 5. Change the plates every 30 min to ensure that embryos can be collected, prepared, and injected before the germ cells form. For an optimal injection workflow, flies should be laying about 200 eggs every 30 min. 1. Change the plate after 30 min laying, and remove the yeast and paper square. Set a timer for 2 min. Add bleach solution directly onto the plate and incubate for 2 min. Wear a lab coat to protect clothing from bleach. 2. Assemble the filtration apparatus with a fresh membrane filter as shown in Fig. 1. Clamp the apparatus together. Add water and filter through to wet the membrane. Start the tap and open the screw on the sidearm flask. After exactly 2 min, tip the bleach from the plates onto the filter. Close the screw just until the liquid goes through, and then open it again, to avoid damaging embryos. 3. Add water to the plates and filter in the same way. Add water to the filter and filter through. Always be aware that too much suction will damage embryos: open the screw on the sidearm flask as soon as the liquid goes through the membrane. Remove 3.3. Preparation of Flies for Egg Laying 3.4. Dechorionation, Lining Up, and Dessi- cation of Embryos
- 23. Transgenesis in Drosophila melanogaster 9 the filter holder and filter from the apparatus; dry excess liquid with paper from underneath. Wash the glass cup to remove embryos sticking to the sides, so that they do not get collected the next time around (see Note 10). 4. Line up embryos. Use a fine paintbrush with a few hairs or a dissection needle to line up embryos in rows in the same anterior-posterior orientation. Leave a small space between embryos as shown in Fig. 2a. Aim for a regular line. The neater the line, the smoother and faster the subsequent injec- tion. Each row can be up to 20 mm long, to fit on a coverslip. Do not line up for longer than 20 min, to ensure that embryos are not too old. With practice, it should be possible to line up 100–200 embryos in 20 min, making several rows of about 60–80 embryos each. 5. Make a line of embryo glue on the edge of a coverslip with a Pasteur pipette and allow to dry for 30 s. Using forceps, very gently touch the line of embryos with the glued edge to pick them up. Take care not to damage the embryos at this step (see Note 6). Turn the cover slip and put it on a glass slide with a drop of water to stick the coverslip to the slide as shown in Fig. 2b. Fig. 2. (a) Line of embryos. (b) Embryos on slide. Place the cover slip with the line of embryos perpendicular to the long edge of the slide as shown.A drop of water between slide and cover slip is sufficient to prevent movement during injection.
- 24. 10 Ringrose 6. Dry the embryos by placing the slide in a large (150 mm) closed petri dish containing self-indicating orange silica gel. The silica gel crystals must be orange. If they are not, replace them with fresh ones. Incubate at 18°C for 15–20 min. The drying time is critical (see Note 11). After drying, cover the line of embryos with Halocarbon oil. This prevents further drying but allows exchange of air. Begin injection. Make sure the needle is mounted and the injection apparatus is ready to start injection immediately after drying (see Subheading 3.5). 1. Switch on the femtojet and allow to warm up (about 5 min). Set the pressure (pi) to 500 hPa, and injection time to 0.5 s. 2. Mount the needle: Remove the needle holder from the micro- manipulator and remove the old needle if necessary. Using a microloader tip, load 2–3 mL of DNA into the new needle, taking care to avoid air bubbles. Mount the new needle into the holder. Mount the holder into the micromanipulator (see Note 12). 3. Place the slide with embryos onto the microscope stage, and use the micromanipulator to position the needle so it is in the centre of the field. Check that the posterior ends of the embryos are facing the needle. If not, raise the needle and turn the slide around, taking care not to damage the needle. 4. Move the embryos away from the needle. Clean the needle and check that a bubble of liquid of the correct size comes out, as shown in Fig. 3a. If the bubble is too small, increase the pressure (pi) but do not exceed 1,000 hPa. If the bubble is still too small, change the needle. 3.5. Microinjection of Embryos Fig. 3. (a) Testing the needle. Position the needle close to, but not touching, the row of embryos. Press the ‘clean’ button. A bubble of approximately the size shown should emerge. Note, when pressing the ‘inject’ button, the bubble will be almost undetectable. (b) Injecting. Insert the needle into the embryo as shown. Press ‘inject’.A small transient movement should be visible.
- 25. Transgenesis in Drosophila melanogaster 11 5. Position embryos and needle. In most setups, the needle is brought to a suitable position using the micromanipulator and is then fixed at that position. The embryos are injected by moving the microscope stage. Use the micromanipulator to position the needle so that it is in the centre of the field of view. Now move the embryos until the needle touches the posterior end of an embryo at its outermost point. This can be tricky and requires some practice. Focus sharply on the outermost posterior point of one embryo and use the micro- manipulator to bring the tip of the needle into the same focal plane. The needle should be perpendicular to the point of penetration. From this point on, the needle should no longer be moved. Now by moving the microscope stage, insert the needle into the outer membrane and through, so it just enters the inner membrane, as shown in Fig. 3b. A short sharp movement works best. 6. Inject. The drop of injected liquid should be visible as a very small movement, like a small pale cloud transiently appearing in the cytoplasm (see Fig. 3b). If a large pale spot remains, decrease the injection pressure. Note the desiccation state of the embryos: If they are too dry they will deform under the pressure of the needle. If they are insufficiently dried, they will leak cytoplasm. Adjust the drying time in the next round if necessary (see Note 13). Inject the row of embryos, clean- ing the needle regularly. Inject only embryos that have not yet formed pole cells, as shown in Fig. 4. Leave out embryos that are too old (see Note 14). Inject 50–100 embryos per Fig. 4. Age of embryos (see Note 14).
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- 27. XLV. COWHERD. 1763–1816. IN any history of Vegetarianism it is impossible to omit record of the lives and labours of the institutors of a religious community who, in establishing humane dietetics as an essential condition of membership, may well claim the honourable title of religious reformers, and to whom belongs the singular merit of being the first and only founders of a Christian church who have inculcated a true religion of life as the basis of their teaching. William Cowherd, the first founder of this new conception of the Christian religion, which assumed the name of the “Bible Christian Church,” was born at Carnforth, near Lonsdale, in 1763. His first appearance in public was as teacher of philology in a theological college at Beverley. Afterwards, coming to Manchester, he acted as curate to the Rev. J. Clowes, who, while remaining a member of the Established Church, had adopted the theological system of Swedenborg. Cowherd attached himself to the same
- 28. mystic creed, and he is said to be one of the few students of him who have ever read through all the Latin writings of the Swedish theologian. He soon resigned his curacy, and for a short time he preached in the Swedenborgian temple in Peter Street. There he seems not to have found the freedom of opinion and breadth in teaching he had expected, and he determined to propagate his own convictions, independently of other authority. In the year 1800 he built, at his own expense, Christ Church, in King Street, Salford—the first meeting-place of the reformed church.[261] His extraordinary eloquence and ability, as well as earnestness of purpose, quickly attracted a large audience, and may well have brought to recollection the style and matter of the great orator of Constantinople of the fourth century. One characteristic of his Church—perhaps unique at that time—was the non-appropriation of sittings. Another unfashionable opinion held by him was the Pauline one of the obligation upon Christian preachers to maintain themselves by some “secular” labour, and he therefore kept a boarding school, which attained extensive proportions. In this college some zealous and able men, who afterwards were ordained by him to carry on a truly beneficent ministry, assisted in the work of teaching, of whom the names of Metcalfe, Clark, and Schofield are
- 29. particularly noteworthy. Following out the principles of their Master, two of them took degrees in medicine, and gained their living by that profession. The Principal himself built an institute, connected with his church in Hulme, where, more recently, the late Mr. James Gaskill presided, who, at his death, left an endowment for its perpetuation as an educational establishment. It was in the year 1809 that Cowherd formally promulgated, as cardinal doctrines of his system, the principle of abstinence from flesh-eating, which, in the first instance, he seems to have derived from “the medical arguments of Dr. Cheyne and the humanitarian sentiments of St. Pierre.” He died not many years after this formal declaration of faith and practice, not without the satisfaction of knowing that able and earnest disciples would carry on the great work of renovating the religious sentiment for the humanisation of the world. Of those followers not the least eminent was Joseph Brotherton, the first M.P. for Salford, than which borough none has been more truly honoured by the choice of its legislative representative. A printing press had been set up at the Institution, and, after the death of the Master, his Facts Authentic in Science and Religion towards a New Foundation of the Bible, under which title he had
- 30. collected the most various matter illustrative of passages in the Bible, and in defence of his own interpretation of them, was there printed. It is, as his biographer has well described it, “a lasting memorial of his wide reading and research—travellers, lawyers, poets, physicians, all are pressed into his service— the whole work forming a large quarto common- place book filled with reading as delightful as it is discursive. Some of his minor writings have also been printed. He was, besides his theological erudition, a practical chemist and astronomer, and he caused the dome of the church in King Street to be fitted up for the joint purposes of an observatory and a laboratory. His microscope is still preserved in the Peel Park Museum. His valuable library, which at one time was accessible to the public on easy terms, is now deposited in the new Bible Christian Church in Cross Lane. The books collected exhibit the strong mind which brought them together for its own uses. This library is the workshop in which he wrought out a new mode of life and a new theory of doctrine— with these instruments he moulded minds like that of Brotherton, and so his influence has worked in many unseen channels.” He died in 1816, and is buried in front of his chapel, in King Street, Salford.[262]
- 31. XLVI. METCALFE. 1788–1862. AMONGST the immediate disciples of the founder of the new community, the most active apostle of the principles of Vegetarianism, William Metcalfe, to whom reference has been already made, claims particular notice. Born at Orton in Westmoreland, after instruction in a classical school kept by a philologist of some repute, he began life as an accountant at Keighley, in Yorkshire. His leisure hours were devoted to mental culture, both in reading and in poetic composition. Converted by Cowherd in 1809, in the twenty-first year of his age, he abandoned the flesh diet, and remained to the end a firm believer in the truths of “The Perfect Way.” In the year following he married the daughter of the Rev. J. Wright who was at the head of the “New Church” at Keighley, and whom he assisted as curate. His wife, of highly-cultured mind, equally with himself was a persistent follower of the reformed mode of living. Sharing the experiences of many
- 32. other dietary reformers, the young converts encountered much opposition from their family and friends, who attempted at one moment ridicule, at another dissuasion, by appealing to medical authority. Unmoved from their purpose, they continued unshaken in their convictions. “They assured me,” he writes at a later period, “that I was rapidly sinking into a consumption, and tried various other methods to induce me to return to the customary dietetic habits of society; but their efforts proved ineffectual. Some predicted my death in three or four months; and others, on hearing me attempt to defend my course, hesitated not to tell me I was certainly suffering from mental derangement, and, if I continued to live without flesh-food much longer, would unquestionably have to be shut up in some insane asylum. All was unavailing. Instead of sinking into consumption, I gained several pounds in weight during the first few weeks of my experiment. Instead of three or four months bringing me to the silent grave, they brought me to the matrimonial altar. “She [his wife] fully coincided with me in my views on vegetable diet, and, indeed, on all other important points was always ready to defend them to the best of her ability— studied to show our acquaintances, whenever they paid us a visit, that we could live, in every rational enjoyment, without the use of flesh for food. As she was an excellent cook, we were never at a loss as to what we should eat. We commenced housekeeping in January, 1810, and, from that date to the present time, we have never had a pound of flesh-meat in our dwelling, have never patronised either slaughter-houses or spirit shops. “When, again, in the course of time we were about to be blessed with an addition to our family, a renewed effort was
- 33. made. We were assured it was impossible for my wife to get through her confinement without some more strengthening food. Friends and physicians were alike decided upon that point. We were, notwithstanding, unmoved and faithful to our principles. Next we were told by our kind advisers that the little stranger could not be sufficiently nourished unless the mother could eat a little ‘meat’ once a day; or, if not that, drink a pint or half a pint of ale daily. To both proposals my wife turned a deaf ear; and both she and the child did exceedingly well.[263] It may be proper to add here [remarks the biographer], that the ‘little stranger’ above referred to is the author of this Memoir,—that he is in the fifty-sixth year of his age, that he has never so much as tasted animal food, nor used intoxicating drinks of any kind, and that he is hale and hearty.” These experiences, it is scarcely necessary to remark, in the lives of followers of reformed dietetics, have been not seldom repeated. In the Academy of Sciences, instituted by Dr. Cowherd, Metcalfe was invited to assume the direction of the “classical” department (1811). In the same year he took “Orders,” and, at the solicitation of the secessionists from the Swedenborgian Communion (which, with some inconsistency, seems to have looked with indifference, or even dislike, upon the principles of akreophagy), he officiated at Adingham, in Yorkshire. By the voluntary aid of one of his admirers a church was built, to which was added a commodious school-room. He then resigned
- 34. his position under Dr. Cowherd, and opened a grammar school in Adingham, where he was well supported by his friends. The United States of America, however, was the field to which he had long been looking as the most promising for the mission work to which he had devoted himself; and in this hope he had been sustained by his Master. In the spring of 1817 a company of forty-one persons, members of the Bible Christian community, embarked at Liverpool for Philadelphia, They comprised two clerics—W. Metcalfe and Jas. Clark—twenty other adults, and nineteen children. Of this band only a part were able to resist the numerous temptations to conformity with the prevalent social practices; and the vast distances which separated the leaders from their followers were almost an insuperable bar to sympathy and union. Settling in Philadelphia—for them at least a name of real significance—Metcalfe supported his family by teaching, while performing the duties of his position as head of the faithful few who formed his church. His day-school, which was attended by the sons of some of the leading people of the city, proved to be pecuniarily successful until the appearance of yellow fever in Philadelphia, which broke up his establishment and involved him in great difficulties; for upon his school he depended entirely
- 35. for his living. He had many influential friends, who tempted him, at this crisis of his fortunes, with magnificent promises of support, if only he would desert the cause he had at heart—the propagandism of a religion based upon principles of true temperance and active goodness. Both moral and physical superiority pointed him out as one who could not fail to bring honour to any undertaking, and, had he sacrificed conviction to interest, he might have greatly advanced his material prospects. All such seductions he firmly resisted. Meanwhile, through the pulpit, the schoolroom, and, more widely, through the newspapers, he scattered the seeds of the gospel of Humanity. But the spirit of intolerance and persecution, of self- seeking religionism, and of rancorous prejudice, was by no means extinct even in the great republic, and the (so-called) “religious” press united to denounce his humane teaching as well as his more liberal theology. Nor did some of his more unscrupulous opponents hesitate, in the last resort, to raise the war-cry of “infidel” and “sceptic.” These assailants he treated with contemptuous silence; but the principle of moral dietetics he defended in the newspapers with ability and vigour. In 1821 he published an essay on Abstinence from the Flesh of Animals, which was freely and extensively circulated. For
- 36. several years his missionary labours appear to have been unproductive. In the year 1830 he made two notable converts—Dr. Sylvester Graham, who was at that time engaged as a “temperance” lecturer, and was deep in the study of human physiology; and Dr. W. Alcott. Five years later, the Moral Reformer was started as a monthly periodical, which afterwards appeared under the title of the Library of Health. In 1838–9 the Graham Journal was also published in Boston, and scientific societies were organised in many of the New England towns. The Bible was largely appealed to in the controversy, and a sermon of Metcalfe’s had an extensive circulation through the United States. With all this controversy upon his hands, he was far from neglecting his private duties, and, in fact, his health was over-taxed in the close and constant work in the schoolrooms, overcrowded and ill-ventilated as they were. In the day and night school he was constantly employed, during one half of the year, from eight in the morning until ten at night; and Sunday brought him no remission of labour. In the propagandism of his principles through the press he was not idle. The Independent Democrat, and, in 1838, the Morning Star, was printed and published at his own office—by which latter journal, in spite of the promise of support from political
- 37. friends, he was a pecuniary loser to a large amount. The Temperance Advocate, also issued from his office, had no better success. Several years earlier, about 1820, it is interesting to note, he had published a tract on The Duty of Abstinence from all Intoxicating Drinks; and the founder of the Bible Christian Church in America can claim the merit of having been the first systematically to inculcate this social reform. In the year 1847 the Vegetarian Society of Great Britain had been founded, of which Mr. James Simpson had been elected the first president. Metcalfe immediately proposed the formation of a like society in the United States. He corresponded with Drs. Graham, Alcott, and others; and finally an American Vegetarian Convention assembled in New York, May 15, 1850. Several promoters of the cause, previously unknown to each other (except through correspondence), here met. Metcalfe was elected president of the Convention; addresses were delivered, and the constitution of the society determined upon. The Society was organised by the election of Dr. William Alcott as president, Rev. W. Metcalfe as corresponding secretary, and Dr. Trall as recording secretary. An organ of the society was started in November, 1850, under the title of The American Vegetarian and Health Journal, and under
- 38. the editorship of Metcalfe. Its regular monthly publication, however, did not begin until 1851. In that year he was selected as delegate to the English Vegetarian Society, as well as delegate from the Pennsylvania Peace Society to the “World’s Peace Convention,” which was fondly supposed to be about to be inaugurated by the Universal Exhibition of that year. The proceedings at the annual meeting of the Vegetarian Society of Great Britain, and the eloquent address, amongst others, of the American representative, are fully recorded in the Vegetarian Messenger for 1852. On this occasion Joseph Brotherton, M.P. presided. Two years later he suffered the irreparable loss of the sympathising sharer in his hopes for the regeneration of the world. Mrs. Metcalfe died in the seventy-fourth year of her age, having been, during forty-four years, a strict abstinent. Her loss was mourned by the entire Vegetarian community. By far the larger part of the matter, as well as the expenses of publication, of the American Vegetarian, was supplied by the editor, and, being inadequately supported by the rest of the community, the managers were forced to abandon its further publication. The last volume appeared in 1854. It has been succeeded in later times, under happier
- 39. circumstances, by the Health Reformer which is still in existence. In 1855 Metcalfe received an invitation to undertake the duties attached to the mother church at Salford. Leaving his brother-in-law in charge of the church in Philadelphia, he embarked for England once more, and the most memorable event, during his stay in this country, was the deeply and sincerely lamented death of Joseph Brotherton, who for twenty years had represented Salford in the Legislature, and whose true benevolence had endeared him to the whole community. Metcalfe was chosen to preach the funeral eulogy, which was listened to by a large number of Members of Parliament and municipal officers, and by an immense concourse of private citizens. Returning to America soon afterwards, at the urgent request of his friends in Philadelphia, he was, in 1859, elected to fill the place of President vacated by Dr. Alcott, whose virtues and labours in the cause he commemorated in a just eulogy. His own death took place in the year 1862, in the seventy-fifth year of his age, caused by hemorrhage of the lungs, doubtless the effect of excessive work. His end, like his whole interior if not exterior life, was, in the best meaning of a too conventional expression, full of peace and of hope. His best panegyric is to be found
- 40. in his life-work; and, as the first who systematically taught the truths of reformed dietetics in the “New World,” he has deserved the unceasing gratitude of all sincere reformers in the United States, and, indeed, throughout the globe. By all who knew him personally he was as much loved as he was esteemed, and the newspapers of the day bore witness to the general lamentation for his loss.[264]
- 41. XLVII. GRAHAM. 1794–1851. AS an exponent of the physiological basis of the Vegetarian theory of diet, in the most elaborate minuteness, the author of Lectures on the Science of Human Life has always had great repute amongst food reformers both in the United States and in this country. Collaterally connected with the ducal house of Montrose, his father, a graduate of Oxford, emigrated to Boston, U.S., in the year 1718. He must have attained an advanced age when his seventeenth child, Sylvester, was born at Suffield, in Connecticut. Yet he seems to have been of a naturally dyspeptic and somewhat feeble constitution, which was inherited by his son, whose life, in fact, was preserved only by the method recommended by Locke—free exposure in the open air. During several years he lived with an uncle, on whose farm he was made to work with the labourers. In his twelfth year he was sent to a school in New York, and at fourteen he was set for a short time to
- 42. learn the trade of paper-making. “He is described as handsome, clever, and imaginative. ‘I had heard,’ he says, ‘of noble deeds, and longed to follow in the field of fame.’ Ill health soon obliged his return to the country, and at sixteen symptoms of consumption appeared. Various occupations were tried until the time, when about twenty years of age, he commenced as a teacher of youth, proving highly successful with his pupils. Again ill-health obliged the abandonment of this pursuit.”[265] At the age of thirty-two he married, and soon after became a preacher in the Presbyterian Church. Deeply interested in the question of “Temperance,” he was invited to lecture for that cause by the Pennsylvania Society (1830). He now began the study of physiology and comparative anatomy, in which his interest was unremitting. These important sciences were used to good effect in his future dietetic crusade. At this time he came in contact with Metcalfe, by whom he was confirmed in, if not in the first instance converted to, the principles of radical dietary reform. “He was soon led to believe that no permanent cure for intemperance could be found, except in such change of personal and social customs as would relieve the human being from all desire for stimulants. This idea he soon applied to medicine, so that the prevention and cure of disease,
- 43. as well as the remedy for intemperance, were seen to consist mainly in the adoption of correct habits of living, and the judicious adaptation of hygienic agencies. These ideas were elaborated in an Essay on the Cholera (1832), and a course of lectures which were delivered in various parts of the country, and subsequently published under the title of Lectures on the Science of Human Life (2 vols., Boston, 1839). This has been the leading text-book of all the dietetic and nearly all the health reformers since.”[266] The Science of Human Life is one of the most comprehensive as well as minute text books on scientific dietetics ever put forth. If it errs at all, it errs on the side of redundancy—a feature which it owes to the fact that it was published to the world as it was orally given. It therefore well bears condensation, and this has been judiciously done by Mr. Baker, whose useful edition is probably in the hands of most of our readers. Graham was also the author of a treatise on Bread and Bread-Making, and “Graham bread” is now universally known as one of the most wholesome kinds of the “staff of life.” Besides these more practical writings, for some time before his death he occupied his leisure in the production of a Philosophy of Sacred History, the characteristic idea of which seems to have been to
- 44. harmonise the dogmas of the Jewish and Christian Scriptures with his published views on physiology and dietetics. He lived to complete one volume only (12mo.), which appeared after his death. Tracing the history of Medicine from the earlier times, and its more or less of empiricism in all its stages, Graham discovers the cause of a vast proportion of all the egregious failure of its professors in the blind prejudice which induces them to apply to the temporary cure, rather than to the prevention, of disease. As it was in its first barbarous beginning, so it has continued, with little really essential change, to the present moment:— “Everything is done with a view to cure the disease, without any regard to its cause, and the disease is considered as the infliction of some supernatural being. Therefore, in the progress of the healing art thus far, not a step is taken towards investigating the laws of health and the philosophy of disease. “Nor, after Medicine had received a more systematic form, did it apply to those researches which were most essential to its success, but, like religion, it became blended with superstitions and absurdities. Hence, the history of Medicine, with very limited exceptions, is a tissue of ignorance and error, and only serves to demonstrate the absence of that knowledge upon which alone an enlightened system of Medicine can be founded, and to show to what extent a noble art can be perverted from its capabilities of good to almost unmixed evil by the ignorance, superstition, and cupidity of
- 45. men. In modern times, anatomy and surgery have been carried nearly to perfection, and great advance has been made in physiology. The science of human life has been studied with interest and success, but this has been confined to the few, while even in our day, and in the medical profession itself, the general tendency is adverse to the diffusion of scientific knowledge. “The result is, that men prodigally waste the resources as if the energies of life were inexhaustible; and when they have brought on disease which destroys their comforts, they fly to the physician, not to learn by what violation of the laws of life they have drawn the evil upon themselves, and by what means they can avoid the same; but, considering themselves visited with afflictions which they have in no manner been concerned in causing, they require the physician’s remedies, by which their sufferings may be alleviated. In doing this, the more the practice of the physician conforms to the appetites of the patient, the greater is his popularity and the more generously is he rewarded. “Everything, therefore, in society tends to confine the practising physician to the department of therapeutics, and make him a mere curer of disease; and the consequence is, that the medical fraternity have little inducement to apply themselves to the study of the science of life, while almost everything, by which men can be corrupted, is presented to induce them to become the mere panderers of human ignorance and folly; and, if they do not sink into the merest empiricism, it is owing to their own moral sensibility rather than to the encouragement they receive to pursue an elevated scientific professional career. “Thus the natural and acquired habits of man concur to divert his attention from the study of human life, and hence
- 46. he is left to feel his way to, or gather from what he calls experience, all the conclusions which he embraces. It has been observed that men, in their (so-called) inductive reasonings deceive themselves continually, and think that they are reasoning from facts and experience, when they are only reasoning from a mixture of truth and falsehood. The only end answered by facts so incorrectly apprehended is that of making error more incorrigible. Nothing, indeed, is so hostile to the interests of Truth as facts incorrectly observed. On no subjects are men so liable to misapprehend facts, and mistake the relation between cause and effect, as on that of human life, health, and disease.” By the opponents of dietetic reform it has been pretended that climate, or individual constitution, must determine the food proper for nations or individuals:— “We have been told that some enjoy health in warm, and others in cold climates some on one kind of diet, and under one set of circumstances, and some under another; that, therefore, what is best for one is not for another; that what agrees well with one disagrees with another; that what is one man’s meat is another man’s poison; that different constitutions require different treatment; and that, consequently, no rules can be laid down adapted to all circumstances which can be made a basis of regimen to all. “Without taking pains to examine circumstances, people consider the bare fact that some intemperate individuals reach old age evidence that such habits are not unfavourable to life. With the same loose reasoning, people arrive at conclusions equally erroneous in regard to nations. If a tribe, subsisting on vegetable food, is weak, sluggish, and destitute
- 47. of courage and enterprise, it is concluded that vegetable food is the cause. Yet examination might have shown that causes fully adequate to these effects existed, which not only exonerated the diet, but made it appear that the vegetable diet had a redeeming effect, and was the means by which the nation was saved from a worse condition. “The fact that individuals have attained a great age in certain habits of living is no evidence that those habits are favourable to longevity. The only use which we can make of cases of extraordinary old age, is to show how the human constitution is capable of sustaining the vital economy, and resisting the causes which induce death. “If we ask how we must live to secure the best health and longest life, the answer must be drawn from physiological knowledge; but if we ask how long the best mode of living will preserve life, the reply is, Physiology cannot teach you that. Probably each aged individual has a mixture of good and bad habits, and has lived in a mixture of favourable and unfavourable circumstances. Notwithstanding apparent diversity, there is a pretty equal amount of what is salutary in the habits and circumstances of each. Some have been ‘correct’ in one thing, some in another. All that is proved by instances of longevity in connexion with bad habits is, that such individuals are able to resist causes that have, in the same time, sent thousands of their fellow-beings to an untimely grave; and, under a proper regimen, they would have sustained life, perhaps, a hundred and fifty years. “Some have more constitutional [or inherited] powers to resist the causes of disease than others, and, therefore, what will destroy the life of one may be borne by another a long time without any manifestations of immediate injury. There are, also, constitutional peculiarities, but these are far more rare than is generally supposed. Indeed, such may, in almost every case, be overcome by a correct regimen. So far as the
- 48. general laws of life and the application of general principles of regimen are considered, the human constitution is one: there are no constitutional differences which will not yield to a correct regimen, and thus improve the individual. Consequently, what is best for one is best for all.... Some are born without any tendency to disease while others have the predisposition to particular diseases of some kind. But differences result from causes which man has the power to control, and it is certain that all can be removed by conformity to the laws of life for generations, and that the human species can be brought to as great uniformity, as to health and life, as the lower animals.” With Hufeland, Flourens, and other scientific authorities, he maintains that:— “Physiological science affords no evidence that the human constitution is not capable of gradually returning to the primitive longevity of the species. The highest interests of our nature require that youthfulness should be prolonged. And it is as capable of being preserved as life itself, both depending on the same conditions. If there ever was a state of the human constitution which enabled it to sustain life [much beyond the present period], that state involved a harmony of relative conditions. The vital processes were less rapid and more complete than at present, development was slower, organisation more perfect, childhood protracted, and the change from youth to manhood took place at a greater remove from birth. Hence, if we now aim at long life, we can secure our object only by conformity to those laws by which youthfulness is prolonged.” As for the omnivorousness of the human animal: —
- 49. The ourang-outang, on being domesticated, readily learns to eat animal food. But if this proves that animal to be omnivorous, then the Horse, Cow, Sheep, and others are all omnivorous, for everyone of them is easily trained to eat animal food. Horses have frequently been trained to eat animal food,[267] and Sheep have been so accustomed to it as to refuse grass. All carnivorous animals can be trained to a vegetable diet, and brought to subsist upon it, with less inconvenience and deterioration than herbivorous or frugivorous animals can be brought to live on animal food. Comparative anatomy, therefore, proves that Man is naturally a frugivorous animal, formed to subsist upon fruits, seeds, and farinaceous vegetables.[268] The stimulating, or alcoholic, property of flesh produces the delusion that it is, therefore, the most nourishing:— “Yet by so much as the stimulation exceeds that which is necessary for the performance of the functions of the organs, the more does the expenditure of vital powers exceed the renovating economy; and the exhaustion which succeeds is commensurate with the excess. Hence, though food which contains the greatest proportion of stimulating power causes a feeling of the greatest strength, it also produces the greatest exhaustion, which is commensurately importunate for relief; and, as the same food affords such by supplying the requisite stimulation, their feelings lead the consumers to believe that it is most strengthening.... Those substances, the stimulating power of which is barely sufficient to excite the digestive organs in the appropriation of nourishment, are most conducive to vital welfare, causing all the processes to be most perfectly performed, without any unnecessary expenditure, thus contributing to health and longevity.
- 50. “Flesh-meats average about thirty-five per cent of nutritious matter, while rice, wheat, and several kinds of pulse (such as lentils, peas, and beans), afford from eighty to ninety-five per cent; potatoes afford twenty-five per cent of nutritious matter. So that one pound of rice contains more nutritious matter than two pounds and a half of flesh meat; three pounds of whole meal bread contain more than six pounds of flesh, and three pounds of potatoes more than two pounds of flesh.” That the human species, taken in its entirety, is no more carnivorous de facto than it could be de jure, is apparent on the plain evidence of facts. In all countries of our Globe, with the exception of the most barbarous tribes, it is, in reality, only the ruling and rich classes who are kreophagist. The Poor have, almost everywhere, but the barest sufficiency even of vegetable foods:— “The peasantry of Norway, Sweden, Denmark, Germany, Turkey, Greece, Italy, Switzerland, France, Spain, England, Scotland, Ireland, a considerable portion of Prussia, and other parts of Europe subsist mainly on non-flesh foods. The peasantry of modern Greece [like those of the days of Perikles] subsist on coarse brown bread and fruits. The peasantry in many parts of Russia live on very coarse bread, with garlic and other vegetables, and, like the same class in Greece, Italy, &c., they are obliged to be extremely frugal even in this kind of food. Yet they are [for the most part] healthy, vigorous, and active. Many of the inhabitants of Germany live mainly on rye and barley, in the form of coarse bread. The potato is the principal food of the Irish peasantry, and few portions of the human family are more healthy,
- 51. athletic, and active, when uncorrupted by intoxicating substances [and, it may be added, when under favourable political and social conditions]. But alcohol, opium, &c. [equally with bad laws] have extended their blighting influence over the greater portion of the world, and nowhere do these scourges so cruelly afflict the self-devoted race as in the cottages of the poor, and when, by these evils and neglect of sanitation, &c., diseases are generated, sometimes epidemics, we are told that these things arise from their poor, meagre, low, vegetable diet. Wherever the various sorts of intoxicating substances are absent, and a decent degree of cleanliness is observed, the vegetable diet is not thus calumniated. “That portion of the peasantry of England and Scotland who subsist on their barley and oatmeal bread, porridge, potatoes, and other vegetables, with temperate, cleanly habits [and surroundings], are able to endure more fatigue and exposure than any other class of people in the same countries. Three-fourths of the whole human family, in all periods of time [excepting, perhaps, in the primitive wholly predatory ages] have subsisted on non-flesh foods, and when their supplies have been abundant, and their habits in other respects correct, they have been well nourished.” That the sanguinary diet and savagery go hand in hand, and that in proportion to the degree of carnivorousness is the barbarous or militant character of the people, all History, past and present, too clearly testifies. Nor are the carnivorous tribes conspicuous by their cruel habits only:— “Taking all flesh-eating nations together, though some, whose other habits are favourable, are, comparatively, well-
- 52. formed, as a general average they are small, ill-formed races; and taking all vegetable-eating nations, though many, from excessive use of narcotics, and from other unfavourable circumstances, are comparatively small and ill-formed, as a general average they are much better formed races than the flesh-eaters.[269] It is only among those tribes whose habits are temperate, and who subsist on the non-flesh diet, that the more perfect specimens of symmetry are found. “Not one human being in many thousands dies a natural death. If a man be shot or poisoned we say he dies a violent death, but if he is ill, attended by physicians, and dies, we say he dies a ‘natural’ death. This is an abuse of language—the death in the latter case being as truly violent as if he had been shot. Whether a man takes arsenic and kills himself, or by small doses or other means, however common, gradually destroys life, he equally dies a violent death. He only dies a natural death who so obeys the laws of his nature as by neither irritation nor intensity to waste his energies, but slowly passes through the changes of his system to old age, and falls asleep in the exhaustion of vitality.”[269] With Flourens he adduces a number of instances both of individuals and of communities who have attained to protracted ages by reason of a pure diet. He afterwards proceeds to prove from comparative physiology and anatomy, and, in particular, from the conformation of the human teeth and stomach (which, by an astounding perversion of fact, are sometimes alleged to be formed carnivorously, in spite of often-repeated scientific authority, as well as of common observation), the natural frugivorous character of the human species, and he quotes
- 53. Linné, Cuvier, Lawrence, Bell, and many others in support of this truth.[270]
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