Table Of ContentAnimalTransgenesisandCloning.Louis-MarieHoudebine
Copyright(cid:182)2003JohnWiley&Sons,Ltd.
ISBNs:0-470-84827-8(HB);0-470-84828-6(PB)
Animal Transgenesis
and Cloning
Animal Transgenesis
and Cloning
Louis-Marie Houdebine
Institut National de la Recherche Agronomique,
Jouy en Josas, France
Translated by
Louis-Marie Houdebine, Christine Young,
Gail Wagman and Kirsteen Lynch
FirstpublishedinFrenchasTransgene`seAnimaleetClonage#2001Dunod,Paris
TranslatedintoEnglishbyLouis-MarieHoudebine,ChristineYoung,GailWagmanand
KirsteenLynch.
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Contents
Introduction ix
AbbreviationsandAcronyms xiii
1 From the gene to the transgenic animal 1
1.1 Genome composition 1
1.2 Gene structure 4
1.3 The number of genes in genomes 7
1.4 The major techniques of genetic engineering 13
1.4.1 Genecloning 13
1.4.2 DNAsequencing 14
1.4.3 Invitrogeneamplification 14
1.4.4 Geneconstruction 14
1.4.5 Genetransferintocells 16
1.5 The systematic description of genomes 21
1.6 Classical genetic selection 26
1.7 Experimental mutation in genomes 27
1.7.1 Chemicalmutagenesis 27
1.7.2 MutagenesisbyintegrationofforeignDNA 29
1.7.3 Mutagenesisbytransgenesis 30
2 Techniques for cloning and transgenesis 33
2.1 Cloning 33
2.1.1 Themainstepsofdifferentiation 33
2.1.2 Cloningbynucleartransfer 37
2.2 Gene therapy 48
2.2.1 Thegoalsofgenetherapy 48
2.2.2 Thetoolsofgenetherapy 49
2.2.3 Theapplicationsofgenetherapy 52
2.3 Techniques of animal transgenesis 54
2.3.1 Theaimsandtheconceptofanimaltransgenesis 54
2.3.2 Genetransferintogametes 60
vi CONTENTS
2.3.3 Genetransferintoembryos 65
2.3.4 Genetransferviacells 69
2.3.5 Vectorsforgeneaddition 73
2.3.6 Vectorsforgenereplacement 85
2.3.7 Vectorsfortherearrangementof
targetedgenes 90
2.3.8 Targetedintegrationofforeigngenes 97
2.3.9 Non-classicalvectorsfortherecombination
oftargetedgenes 105
2.3.10 Vectorsforgenetrap 106
2.3.11 Vectorsfortheexpressionoftransgenes 116
3 Applications of cloning and transgenesis 137
3.1 Applications of animal cloning 137
3.1.1 Basicresearch 137
3.1.2 Transgenesis 142
3.1.3 Animalreproduction 143
3.1.4 Humanreproduction 144
3.1.5 Therapeuticcloning 144
3.1.6 Xenografting 150
3.2 Applications of animal transgenesis 153
3.2.1 Basicresearch 153
3.2.2 Studyofhumandiseases 154
3.2.3 Pharmaceuticalproduction 159
3.2.4 Xenografting 162
3.2.5 Breeding 163
4 Limits and risks of cloning, gene therapy
and transgenesis 171
4.1 Limits and risks of cloning 173
4.1.1 Reproductivecloninginhumans 173
4.1.2 Reproductivecloninginanimals 175
4.1.3 Therapeuticcloning 176
4.2 Limits and risks of gene therapy 177
4.3 Limits and risks of transgenesis 178
4.3.1 Technicalandtheoreticallimits 178
4.3.2 Biosafetyproblemsinconfinedareas 179
4.3.3 Theintentionaldisseminationoftransgenic
animalsintotheenvironment 181
4.3.4 Therisksforhumanconsumers 184
4.3.5 Transgenesisandanimalwelfare 185
CONTENTS vii
4.3.6 Patentingoftransgenicanimals 187
4.3.7 Transgenesisinhumans 188
ConclusionandPerspectives 191
References 199
Index 217
Introduction
Since the beginning of time, humans have known how to distinguish
living organisms from inanimate objects. Cro-Magnon people and their
descendants were no doubt aware that living beings all had the same
abilitytogrowandmultiplybyrespectingthespecificityofthespecies.It
probably took them longer to understand that heat destroyed living
organisms, whereas the cold, to a certain extent, conserved them.
These very ancient observations have fixed in our minds the notion
thatlivingorganismsarefundamentallydifferentfrominanimatematter.
We now know that living beings are also subject to the laws of thermo-
dynamics, that they are no more than very highly organized matter and
that they only conserve their wholeness below about 1308C.
Wellbeforehavingunderstoodwhatmadeuptheveryessenceofliving
beings, the different human communities learned to make the most of
what they had, sometimes without even realizing it. The existence of
micro-organisms was unknown until the 19th century and yet fermenta-
tion has been carried out for thousands of years in certain foods. Agri-
culture,farmingandmedicinebenefitedfromempiricalobservationsthat
enabled genetic selection and the preparation of medicine, particularly
from plant extracts.
The situation changed radically during the 19th century with the
discovery of the laws of heredity by Gregor Mendel, the theory of
evolutionbyCharlesDarwinandthediscoveryofcells.Theclassification
of living beings has progressively demonstrated their great similarity in
spiteoftheirinfinitediversity.Jean-BaptisteLamarckaswellasCharles
Darwin accumulated observations supporting the theory of evolution.
The two scientists admitted that the surrounding environment had and
continued to have a great influence on the evolution of living beings.
Darwin was the person who most contributed to establishing the idea
thatlivingbeingsmutatedspontaneouslybychanceandtheenvironment
was responsible for conserving only those that were the best adapted to
x INTRODUCTION
the conditions at the time. Mendel determined in what conditions the
traits were transmitted to the progeny, thus establishing the laws of
heredity.
The innumerable observations made possible by the invention of
the microscope in the 17th century revealed the universal existence
ofcellsinalllivingbeings.Theremarkablepropertiesoflivingorganisms
began to be explained: their resemblance, their evolution and their
diversity.
We had to wait until the discovery of the principal molecules that
constitutelivingorganisms(proteins,nucleicacids,lipids,sugarsetc.)to
begintounderstandthechemicalmechanismsthatgoverntheirexistence.
Thetheoriesofthe19thcenturyarenowconfirmedeverydayatthemost
intimateleveloflivingbeings,andinparticularbytheobservationofthe
structure of genes and proteins.
It is now acknowledged that the big bang, which must have occurred
15 billion years ago, was followed by an expansion of matter, which,
when cooling down, progressively and continuously gave way to par-
ticles, atoms, mineral molecules, organic molecules and finally living
organisms. Only the present specific conditions on Earth enable the
highly organized matter of living organisms to survive, proliferate and
evolve.
The discovery of the structure of genes and proteins as well as the
identification of the genetic code about 40 years ago enabled us to
comprehend for the first time what living organisms are and how they
function. Even more, these discoveries have in principle provided
humans with new and powerful means to observe and make use of
certain living species. This has required mastering a certain number of
techniques,whichwegrouptogetherunderthetermgeneticengineering.
From the moment it was known that the structure of DNA directly
determines the structure of proteins, it was in principle possible to
manipulate one or the other by chemical reactions that determine and
modify the structure of genes. This presupposes that the genetic infor-
mation manipulated in this way can be expressed. In practice this is not
possible, and only makes sense if the gene can give rise to the corres-
pondingproteinandiftheproteincanexerciseitsbiochemicalproperties
in the complex context of life. To do so, the isolated and possibly
modified gene can be reintroduced into a cell or a whole organism. It is
for this reason that gene transfer occupies an essential place in modern
biology as well as in biotechnological applications.
INTRODUCTION xi
Intheperiodofonlyafewdecades,theworkofbiologistshaschanged
dramatically. For about a century, biologists had worked essentially
in vivo on whole animals, plants or micro-organisms. This made it
possible to define the role of the principal functions of living organisms,
toidentifyanumberofhormonesetc.Thetraditionalscientificapproach
is based on systematically dividing up problems to try to simplify them
and thus resolve them. Biologists have therefore started to work in cello
with cultured isolated cells. This promising simplification has been
followedbystudiesconductedinvitrousingcellextractsorevenpurified
molecules. The huge quantity of information provided by genome map-
pingandtheircompletesequencingrequiresbiologiststouseotherways
todealwiththeproblems.Thisinformationissovastthatitneedstobe
dealt with in silico by powerful computer processing.
The present situation is particularly promising. Biologists have the
means of knowing all the genetic information of a living organism
throughthecompletesequencingofitsDNA.Itisclearthattheprimary
structureofagenemakesitpossibletopredictthatofthecorresponding
protein. Most often, it only indicates very partially the role of the
protein. Proteins, like genes, are derived from each other during evolu-
tion. Therefore, it is sometimes possible to determine that a protein,
whose structure has been revealed by sequencing its gene, has for
example a kinase activity, by simple structure homology with that of
other proteins known to possess this type of enzymatic activity. The
predictions often stop at this level or never even reach it. The transfer
oftheisolatedgeneinacelloreveninawholeorganismislikelytoreveal
thebiologicalpropertiesofthecorrespondingprotein.Thustheoversim-
plification which the isolation of a gene represents is accompanied by a
return to its natural complex context, which is the living organism.
Hence,biologistsareexperiencingaspectacularlinkbetweentraditional
physiology and molecular biology. This is now referred to as postge-
nomics.
Inthiscontext,transgenesishasanincreasinglyimportantroledespite
allitstheoreticalandtechnicallimits.Thisiswhytransgenesisworkshops
are developing in order to enable researchers to try to determine in vivo
the role of all the genes that are progressively available to them.
Reproductionhasalwaysplayedanessentialroleinthelifeofhumans.
Theythemselvesreproduceofcourseandsometimeswithmoredifficulty
than they would like or in contrast with an excessive prolificacy.
Livestock farming and agriculture are to a great extent based on
reproduction.Inanimals,controllingreproductionhasoccurredprogres-
xii INTRODUCTION
sively. It involved successively favouring mating or not, carrying out
artificial insemination, embryo transfer, in vitro fertilization and finally
cloning.Alltheseoperationsaimessentiallyatincreasingtheefficiencyof
reproduction(forbreedinganimalsinlargenumbers)andatenablingan
effective genetic selection. These techniques are receiving increasing
back-up from the fundamental study of reproduction mechanisms.
The case of cloning does not escape this rule. Cloning animals began
withabiologist’sexperiment.Itwasadoptedbybiotechnologistseagerto
speed up progress in genetics by introgressing the genomes validated by
their very existence as is already the case in plants. In all species, trans-
genesisdependsverymuchoncontrollingreproduction.Thetechniqueof
cloning has shown that it was indeed at the source of a simplification of
genetransferandanextensionofitsuse.Reproductivecloningcould,in
principle, become a new mode of assisted reproduction for the human
species. Therapeutic cloning could in principle help in reprogramming
differentiated cells from a patient in order to obtain organ stem cells to
regenerate defective tissues.
Cloning and transgenesisand thegeneration ofcells forhumantrans-
plants are henceforth very closely associated. Cloning is the opposite of
sexual reproduction, which is accompanied by the reorganization of
genes. The fundamental aim of transgenesis, on the other hand, is to
modify the genetic heritage of an individual or even a species. The
reprogramming of cells concerns the differentiation mechanisms irre-
spective of any genetic modification. This book sets out to give a clear
picture of recent developments in research and its applications in these
three fields. It does not describe the techniques in detail, namely those
used to generate transgenic animals. The readers may find this infor-
mation in other books edited by C.A. Pinkert (2002) and A.R. Clarke
(2002).
Acknowledgements
The author wishes to thank Ms Annie Paglino, Christine Young, Gail
Wagman,KirsteenLynchandMrJoelGalle´ fortheirhelpintheprepar-
ation of this manuscript.
