Journal of Biotechnology 98 (2002) 145 160

www.elsevier.com/locate/jbiotec

The methods to generate transgenic animals and to control

transgene expression
Louis-Marie Houdebine *
Biologie du De6eloppement et Biotechnologies, Institut National de la Recherche Agronomique, 78352 Jouy en Josas Cedex, France
Received 10 July 2001; received in revised form 28 January 2002; accepted 11 February 2002

Abstract
Transgenic animals have been used for years to study gene function and to create models for the study of human
diseases. This approach has become still more justified after the complete sequencing of several genomes. Transgenic
animals are ready to become industrial bioreactors for the preparation of pharmaceuticals in milk and probably in
the future in egg white. Improvement of animal production by transgenesis is still in infancy.
Despite its intensive use, animal transgenesis is still suffering from technical limitations. The generation of
transgenics has recently become easier or possible for different species thanks to the use of transposons or retrovirus,
to incubation of sperm which DNA followed by fertilization by intracellular sperm injection or not and to the use of
the cloning technique using somatic cells in which genes have been added or inactivated. The Cre-LoxP system is
more and more used to withdraw a given sequence from the genome or to target the integration of a foreign DNA.
The tetracycline system has been improved and can more and more frequently be used to obtain faithful expression
of transgenes. Several tools: RNA forming a triple helix with DNA, antisense RNA including double strand RNA
inducing RNA interference and ribozymes, and also expression of proteins having a negative transdominant effect,
are tentatively being improved to inhibit specifically the expression of host or viral genes.
All these techniques are expected to offer experimenters new and more precise models to study gene function even
in large animals. Improvement of breeding by transgenesis has become more plausible including through the precise
allele replacement in farm animals. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Transgenesis; Gene transfer; Transgene expression

1. Introduction
The discovery of genetic code about 40 years
ago suggested that gene isolation and transfer into
living organisms would become major tools for
* Tel.: +33-1-34-65-25-40; fax: +33-1-34-65-22-41.
E-mail address: houdebine@diamant.jouy.inra.fr (L.-M.
Houdebine).

biologists. The first gene transfer into mouse using

isolated DNA revealed that the generation of
animals stably harboring foreign DNA (Gordon
et al., 1980) and having modified phenotypic
properties (Palmiter et al., 1982) was possible.
These pioneer and fascinating experiments also
revealed some of the limits of transgenesis: the
generation of transgenic animals by gene microinjection appeared laborious; the first transgene un-

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L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145160

expectedly remained very poorly active or inactive; the growth hormone gene induced an overgrowing of the transgenic mice with numerous
physiological side-effects. These observations revealed that the reintegration of an isolated gene
into the genome of an animal may generate complex and unpredictable biological situations.
Direct gene injections were extended to three
others mammals in 1985 (Hammer et al., 1985)
and later to several lower vertebrates and invertebrates. Gene replacement by homologous recombination was achieved in 1989 (Capecchi, 1989).
As many as 5000 mouse genes had been inactivated by this method in 2000.
The better knowledge of gene structure and
function allows the preparation of recombinant
genes having a more predictable expression in
transgenic animals.
It is now well-established that transgenesis is
one of the major tools of biologists to study gene
expression and function. Transgenesis is still going to be used more extensively and systematically
with the identification of all the human genes.
Transgenesis also plays an essential role for applications in the medical and agronomic fields. The
study of human diseases is greatly facilitated by
the generation of transgenic animals mimicking
health disorders or allowing the evaluation of new
pharmaceuticals. Transgenic pigs are expected to
be the source of organs and cells for transplantation to humans. Recombinant proteins of pharmaceuticals interest are being prepared in the milk
of transgenic animals. A few lines or farm animals
having improved breeding properties have been
generated and are under study for human
consumption.
Despite this impressive and growing success,
transgenesis still suffers from many imperfections.
The present paper aims at describing the state of
the art in the animal transgenesis field.

2. The methods to generate transgenic animals

The establishment of stable transgenic animals
implies that the foreign DNA is present in
gametes or one-cell embryos to allow its transmission to progeny. To reach this goal, the foreign

gene can be transferred using different methods

according to animal species.

2.1. DNA microinjection

The direct DNA microinjection into the pronuclei of embryos was the first technique which led
to regular and relatively easy success in mammals.
Essentially the same protocol is followed in
mouse, rat, rabbit, pig, sheep, goat and cow,
however with a decreasing yield of transgenic
animals from mouse to cow. Bovine has a slow
reproduction rate. The number of embryos generated by superovulation is low and the success of
microinjection appeared accessible only if embryos were prepared in vitro after oocyte maturation and fertilization followed by in vitro
development of the microinjected to the blastocyst
stage (Krimpenfort et al., 1991). This method
remains laborious and costly.
In lower vertebrates and invertebrates, pronuclei are not visible gene. Microinjection must
therefore be performed in cytoplasm, using much
larger amounts of DNA. For unknown reasons,
the success of this approach is quite variable from
one species to another. It proved efficient in several fish species and mainly in salmonids (Devlin,
1997). It remains inefficient in the laboratory fish
medaka as well as in xenopus and chicken. In
these species, foreign DNA usually does not integrate into the genome of the animals.
In insects (Drosophila) and worms (Caenorhabditis elegans) (Thierry-Mieg et al., 1997), foreign
DNA is injected into gonad syncytium.

2.2. The use of transposons

In several species, foreign DNA injected in
embryo cytoplasm becomes very rarely integrated
in the genome. This is the case for medaka,
Drosophila, chicken, silk worms etc. In order to
enhance the frequency of integration, several tools
have been implemented. One possibility consists
in generating breaks in host DNA by injecting
low amounts of restriction enzymes. The DNA
repair mechanism restores DNA and integrates
the foreign DNA. This approach proved to be
uneasy to manage. Low concentrations of restric-

L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145160

tion enzyme, have no significant effects whereas

high concentrations are deleterious for the host
genome. The optimum protocol was not found
and this approach has not been retained.
Another possibility consists of using vectors
which have the intrinsic capacity to integrate in a
genome with high efficiency. Retrotransposons
and retrovirus belong to this category.
Transposons are DNA sequences which contain
at least one gene coding for a transposase and
motives located on both ends of the transposons
the role of which is to trigger integration. Transposons sequences are transcribed in RNA, which
drive transposase synthesis. The RNA is retrotranscribed in DNA, which integrates in the multiple sites of the genome under the action of the
transposase. Numerous families of transposons
have been identified.
To become a vector for gene transfer, a transposon must be genetically modified. The transposase gene must be deleted to make space for a
foreign gene and to prevent the recombinant
transposon to disseminate in an uncontrolled
manner. This recombinant vector is unable alone
to integrate into a genome. The exogenous transposase must complement the vector. In practice, a
circular plasmid containing a construct capable of
expressing the transposase gene is injected with
the recombinant vector. This allows the integration of the foreign gene with the vector whereas
the assistant plasmid is rapidly degraded.
Several transposons have been used successfully. Transposon P have used for years to generate transgenic Drosophila (Kayser, 1997). The
mariner
transposon
originally
found
in
Drosophila and adapted to different species is
efficient to transfer gene in medaka (Hackett et
al., 2002), chicken (Shermann et al., 1998) and
mouse (Dupuy et al., 2002).
A recent work showed that transgenic silk
worm expressing the GFP gene could be obtained
using the piggy-Bac transposon (Tamura et al.,
1999).
Transposons are potent and flexible tools which
might be used for gene therapy. They also appear
safe although the mariner transposon seems to be
complemented to some extent by endogenous
transposases and spread at a low rate in the host

147

genome. This is not the case for the piggy-Bac

transposon, which has a narrow spectrum of hosts
and proved to be fully stable over 15 generations
in transgenic silk worms.

2.3. DNA transfer into gametes

One logical approach to generate transgenic
animals may theoretically consist of introducing
foreign DNA in gametes before fertilization.
The incubation of spermatozoa in the presence
of DNA followed by in vivo or in vitro fertilization led to the generation of transgenic mice, fish,
chicken, rabbits, pigs, sheep and cows. However,
the results obtained with this method are inconsistent. The yield of transgenic animals is usually
low and largely unpredictable. Moreover, the integrated DNA is most of the times profoundly
rearranged and no more functional. This phenomena can seemingly be greatly attenuated by selecting the most appropriate ejaculates and by
removing DNAse by repeated washing and addition of DNAse inhibitors (Baccetti and
Spadafora, 2000). This method in its improved
version might become attractive in future for
some species.
In xenopus, gene transfer by spermatozoa was
achieved successfully but on condition to alter
sperm membrane by an incubation with detergent
or by freezing and thawing to allow foreign DNA
to penetrate into the cell. After this first step,
spermatozoa have become inactive and intracellular sperm injection must be performed to obtain
fertilization (Marsh-Armstrong et al., 1999). This
method was applied successfully in mouse but it
appeared no more attractive than the conventional microinjection (Perry et al., 1999). This
elegant protocol is the only available to generate
transgenic xenopus. It might be used for other
species for which microinjection is inefficient.
A quite attractive tool has been recently proposed. Linear DNA containing a functional gene
was bound in a non-covalent manner to a monoclonal antibody, which recognizes a spermatozoa
specific antigen. The spermatozoa were incubated
with the antibody-DNA complex and further used
to fertilize oocyte by in vitro fertilization in
mouse, by artificial insemination in chicken and

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L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145160

by injection into uterine horn in pig. In all

cases, up to 30% of born animals were transgenic.
The transgenes were expressed and transmitted
to progency without any rearrangement. Interestingly the same antibody recognized the same antigen in several mammals including human and in
lower vertebrates. This method might greatly simplify gene addition in animals (Qian et al.,
2001a,b).
Experiments aiming at transferring foreign gene
into sperm precursors either in vivo or in vitro
are in course (for review articles see Baccetti and
Spadafora, 2000). Although encouraging, the results obtained by different groups indicate that
additional studies are required before this approach may become an attractive alternative to
the other methods.
The mechanism of gene transfer into epididymal spermatozoa by injection of a DNA-transfectant complex into testis is under study. This
method permitted the generation of limited number of transgenic animals so far (Sato et al.,
2002). A retroviral vector carrying the b-galactosidase gene was used to infect male germ-line
stem cells. These cells was further transplanted
into the testis of unfertile recipient mice. Approximately 4.5% of progeny from these males were
transgenic and the transgene was transmitted to
progeny and expressed in subsequent generations
(Nagano et al., 2001).
In case, sperm precursors could be cultured and
matured, they could be used not only to add
foreign genes but also to replace host genes by
homologous recombination.
DNA microinjection into oocyte was never or
very rarely followed by the birth of transgenic
animals. Recent experiments showed that retroviral vectors introduced between the zona pellucida
and the plasma membrane of the oocytes can be
implemented to generate transgenic cows (Chan
et al., 1998) and monkeys (Chan et al., 2001).
This success was met with viral particles containing the VSV-G envelope, which is known to recognize membrane phospholipids of all cell types.
The infection was performed at a period when the
nuclear membrane of the oocyte was non-existent.
This greatly enhanced the chance of the recombinant retroviral to reach the host genome and

become integrated. This method is laborious and

less attractive than cloning to generate transgenic
cows. It is the only technique, which allowed the
generation of transgenic primates so far.

2.4. The use of retro6iral 6ectors

Retroviral vectors are under intensive study
to tentatively transfer gene to human somatic
cells and proceed to gene therapy. Similar vectors
have been designed to transfer foreign genes
into mammalian embryonic cells. This approach
proved to be laborious and less efficient than
classical microinjection. A family of vectors
capable of infecting chicken primordial germ cells
and of generating transgenic animals has
been described (Ronfort et al., 1997). Although
laborious this method remains the only, which
allowed repeatedly to transfer foreign gene into
chicken.
A recent unpublished work carried out in D.
Baltimore laboratory has shown that lentiviral
vectors transfer foreign gene with quite high efficiency in one cell embryo. This approach appears
much simpler than microinjection and might become used extensively in future (Lois et al., 2002).

2.5. Gene transfer using embryonic cells

Gene replacement by homologous recombination is performed in routine in bacteria and yeast.
It can be achieved in somatic mammalian cells
although with a relatively poor efficiency. For
unknown reasons, homologous recombination is
more frequent in pluripotent embryonic cells.
This approach is very attractive since it can lead
to specific gene inactivation, to targeted point
mutation in an animal genome or to the replacement of a given gene by a non-related one.
Homologous recombination is a rare event.
Cells in which it occurred must be selected and
further used to generate a living embryo. This
proved to be feasible on condition to use embryonic stem cells capable of forming chimeric embryos after microinjection into blastocysts or
morula (Viville, 1997). Although laborious this
protocol has become popular and genes are frequently inactivated in mouse.

L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145160

Despite repeated efforts, the extension of this

method to species other than mouse failed. This is
clearly due to the fact that the recombined ES
cells have more or less the capacity to participate
to the development of chimeric embryos but that
transmission of the mutation to progeny has been
observed so far only in two mouse lines and
essentially of the 129/SV line (Smithies, 2001). For
years, it was admitted that mouse was the model
for the use of ES cells to mutate genes in a
targeted manner. The systematic lack of success
met in rat, rabbit, chicken, pig, sheep and cow
now inclines to consider that the so-called ES cells
cannot be used for the germinal transmission of a
mutation except in two mouse lines systematic
studies to tentatively identify genes involved in the
two mouse lines are in course. They might contribute to define conditions to use ES cells and
chimeric animals to replace genes in various
species.
It should be mentioned that in a work published recently, it was reported that appropriate
vectors are capable of inducing targeted gene
transfer into Drosphila by homologous recombination (Bernards and Hariharan, 2001).
Two recent studies indicate that chicken cultured primordial germ cells retransferred to embryos can participate to their development and
transmit their genes to progeny (Petitte et al.,
2002). ES like cells from medaka embryo capable
of generating chimaeric animals have also been
described and are potential tools for gene transfer
and targeting in fish (Collodi, 2001).

2.6. Gene transfer by nuclear transfer

In front of the repeated failure met with homologous recombination in ES cells followed by
the formation of chimeric embryos, experimenters
addressed the problem by a quite different
method.
Experiments carried out about 40 years ago
revealed that the nucleus from embryonic cells
experimentally transferred into xenopus enucleated oocytes gave birth to living animals. This
method was extended to sheep about 15 years ago
but the success was restricted to experiments in
which cells used as nuclear donors were taken

149

from early embryos and not previously cultured.

These observations gave credence to the idea that
animal cloning by nuclear transfer was possible
only with nuclei from totipotent or pluripotent
cells.
A systematic study carried out in sheep revealed
that fully differentiated cells as well as embryonic
or foetal cells could successfully give their genes
to generate normal animals. (Wilmut et al., 1997).
The conditions defined for cloning animals starting from differentiated cells was retained soon
after to generate transgenic sheep by gene addition (Schnieke et al., 1997). Gene replacement was
also achieved in sheep (Ayares, 1999; McCreath et
al., 2000) and pig (Dai et al., 2002; Lai et al.,
2002). This experiment is not an easy task. Several
independent problems must be solved to meet the
success. Homologous recombination is less frequent in differentiated cells than in ES cells. In
the experiments described above, ftal fibroblasts
were used to add genes by transfection and replace gene by homologous recombination. These
cells, although of ftal origin, have a limited
number of possible multiplication cycles which
corresponds roughly to the number of passages
required to select clones in which homologous
recombination took place. The culture of the recombined cells must therefore be performed with
a particular care. On the other hand, it has been
repeatedly observed that cells from adults and
cells cultured for long periods of time is a less
efficient material for cloning by nuclear transfer.
The reasons why these phenomena occur are unknown. Mutation of essential genes for embryo
development may occur progressively in living
animals and in cultured cells. Alternatively, chromatin may progressively adopt a less reversible
conformation which reduces the chance of a successful cloning. Interestingly, a recent study
showed that the donor genome in bovine embryos
generated by nuclear transfer from somatic cells is
aberrantly methylated (Kang et al., 2001). This
may have inactivated genes required for embryo
development.
In a recent publication, it was reported that
cloned sheep having knocked-out PrP gene did
not survive after birth (Wells, 2001; Denning et
al., 2001). This illustrates the difficulty of the task.

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L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145160

The cloning approach to knock gene out was

used successfully in mouse with no evidence that
it is less laborious than the classical ES cellchimera method (Readeout et al., 2000).
Gene addition by the cloning technique have
been extended to goat (Reggio et al., 2001) and
cow (Arat et al., 2001; Zakhartchenko et al.,
2001) and pig (Dai et al., 2002). Three groups
obtained cloned pigs independently and by different methods. Gene knock-out was recently
achieved in the pig. Moreover, cloning was successful in the rabbit (Chesne et al., 2002).
This suggests that gene replacement will become a reality in several species other than
mouse in the coming years. Obviously, quite significant progress in the cloning technique and
mainly in the culture of transfected cells to be
used as nuclear donors must be done before regular success can be hoped. Gene addition by the
cloning method is preferred by experimenters
working in ruminants rather than the classical
microinjection technique. It is by no means certain that cloning will be adopted to add gene in
the highly proliferic species (mouse, rat, rabbit
and pig). Microinjection remains presently more
advantageous.

3. The methods to control gene expression

3.1. The reliability of transgene expression

Many transgenes work poorly. Their expression often is very low or not specific of the
promoter added in the gene construct. It is generally admitted that these events result from position effects in chromatin. Enhancers and silencers
from neighbor genes are supposed to activate or
inactivate transgenes. Precise experimental data
support this view. Genomic DNA surrounding a
well-expressed transgene favors the expression of
another transgene when added to it. The phenomena which govern this effect seem however
rather complex. Subtle interactions between the
transgene proper and the genomic DNA sequences seems to influence the expression of the
foreign gene (Cranston et al., 2001).
The stimulation of transgene expression by ge-

nomic enhancers and their extinction by silencers

seem not symmetrical. The second is a phenomenon more frequent and more potent than
the first. Numerous observations indicate that a
transgene is generally poorly expressed (i) when it
contains a cDNA rather than the corresponding
genomic DNA sequence with its introns (ii) when
multiple copies are integrated in the same site (iii)
when a bacterial gene is used. This is reminiscent
of the extinction of transposons and retroviral
genomes. The mechanisms involved in these cases
might also operate to silence transgenes.
Several approaches are possible to reduce ectopic expression and silencing of transgenes.
In eucaryotic genomes, genes and gene clusters
are boardered by DNA regions capable of preventing interactions between neighbor genes.
These sequences which have not yet been well
described are named insulators (Bell et al., 2001).
Some of insulators have a potent silencer effect. This is the case for the 5%HS4 region from
the locus control region (LCR) of the chicken
b-globin locus. This sequence added in duplicate
to transgenes strongly attenuated position effect
(Taboit-Dameron et al., 1999).
Numerous data support the idea that insulators stimulate transgene expression by preventing
the local formation of inactive heterochromatin.
However, it is not presently clear if insulators
taken as a whole have just a silencer effect or if
some of them contain enhancers and must be
considered as chromatin openers.
Most likely, an increasing number of insulators
are going to be described and used in the coming
years. An alternative to the use of identified insulators may consist of utilizing long genomic
DNA fragments ( 100 kb) surrounding the gene
of interest. This genomic material is generally
quite active as transgene. One of the difficulty of
this approach is that long DNA fragment must
be recombined to introduce foreign DNA. Various methods based or homologous recombination in yeast or preferably in bacteria have been
defined to prepare gene constructs with long
DNA fragments (Giraldo and Montoliu, 2001).
In addition to the use of insulators, a certain
number of rules must be respected to avoid low

L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145160

expression of transgenes. When cDNAs rather

than genes are to be used, the addition of a least
one intron before the cDNA is required. The
gene construct must contain as little as possible
of CpG motives specially in the promoter region.
Unexpectedly, house-keeping genes which have
CpG stretches are generally expressed in all cell
types with limited regulation. The same genes are
frequently extinguished when they have become
transgenes (Cohen-Tannoudji et al., 2000). A
chemical synthesis of the cDNA aiming at reducing the number of CpG and at choosing the most
commonly used codons in animal cells may be
recommended, specially for bacterial genes
(Henry et al., 1999). Cryptic splicing sites are
sometimes introduced in gene constructs. This
may lead to the synthesis of truncated and nonfunctional mRNAs. An examination of the vector
sequence on a case by case basis must be done to
avoid these problems. These recommendations
and others have been detailed elsewhere
(Houdebine et al., 2002).
In a certain number of situations, it may be
advantageous to express two cistrons from the
same construct. Two independent transcriptional
units may be cloned in the same vector. Alternatively more compact vectors may be prepared
using internal ribosome entry site (IRES). These
elements are known to allow the translation of
two adjacent cistrons when added between these
coding sequences. Numerous studies have shown
that the action of IRES is somewhat unpredictable. A systematic study revealed that, for
some IRES at least, the terminator codon of the
first cistron must be at about 80 nucleotides of
the IRES to allow an optimal expressing of the
second cistron (and even of the first) (Houdebine
and Attal, 1999).

3.2. The 6ectors to inhibit endogenous gene

expression
Homologous recombination is an excellent way
to suppress the expression of a gene in a whole
animal. This method suffers from several intrinsic
limitations. It is a laborious method not efficient
in all cases. On the other hand, the method implies that the targeted gene is knocked out at the

151

early stage of embryo development. This does not

reflect the subtle regulation to which genes are
normally submitted. Conditional recombination
may be triggered in a given cell type of the
animal and at a given period of its life. This
implies that two LoxP sequences from P1 phage
are initially added to both ends of the targeted
gene.
LoxP sequences can recombine with high efficiency and specificity under the action of the Cre
recombinase from P1 phage. This recombination
eliminates the genomic sequence located between
the two LoxP and knock the gene out. The presence of Cre recombinase induces the LoxP recombination. The enzyme can be synthesized
from plasmids transferred to cells and transiently
expressed. Alternatively, adenoviral vectors harboring a construct expressing Cre recombinase
gene may be injected into a given tissue to induce
local LoxP recombination. Mice harboring a stable transgene containing the Cre recombinase
gene under the control of a tissue specific promoter may be crossed with other transgenic mice
in which targeted introduction of LoxP sequences
has been performed. The inactivation of the gene
induced by LoxP recombination will occur specifically in the tissue in which the promoter is
active. Another level of control can be added to
the system. Fusion genes containing the Cre recombinase and the tamoxifen or RU 486 receptor
sequences have been generated (Wunderlich et al.,
2001) The Cre recombinase activity of these fusion proteins is induced by the administration of
tamoxifen or RU 486 respectively. These systems
offer an additional control of LoxP recombination and of gene knock out. The systems for the
control of transgene expression based on the use
of inducers such tetracycline described below can
also be implemented to tune Cre recombinase
gene expression (Nagy, 2000).
A certain number of mouse lines express the
Cre recombinase gene in specific tissues. They can
be obtained from the laboratories in which they
were prepared or from the Jackson Laboratory
or Charles River Company.
Alternative approaches may be envisaged in
some cases.

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L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145160

3.2.1. The inhibition of endogenous gene

expression by the formation of triple helix
A DNA or RNA region rich in pyrimidine may
form a stable triple helix with a complementary
purine rich DNA or RNA strand. The triple helix
structure specifically prevents transcription and
translation (Upegui-Gonzalez et al., 2000). Although attractive, these methods have never been
proved efficient in transgenic animals. Additional
studies are required to evaluate to which degree
they may really be reliable tools to control expression of endogenous genes.
3.2.2. The inhibition of endogenous expression by
antisense RNAs
Natural mechanisms capable of blocking selectively the expression of endogenous genes are
based on the expression of antisense RNAs which
form hybrids with the targeted mRNAs and prevent their translation. Artificial antisense RNAs
are being used but most of the times with a low
efficiency. It is admitted that this is essentially due
to the fact that the antisense RNAs and the
targeted mRNA sequences have little chance to
interact. Both partners have often secondary
structure and they are associated with proteins
rendering their contact unlikely.
The same is true for the antisense RNAs having
ribozyme activity capable of cleaving the targeted
mRNAs at specific sites. A systematic search of
the accessible sites in targeted mRNAs may be
done by different methods. On the other hand, the
ribozymes may be inserted in one of the loops of
a tRNA or other small RNAs. A recent study
showed that when the ribozyme is associated with
an RNA helicase, any site of an RNA can be
reached and cleaved by the ribozyme (Warashina
et al., 2001).
Numerous experiments have shown that in
plants, in invertebrates and in mammal embryos,
double strand RNAs strongly induce a specific
degradation of the mRNA having the same RNA
sequence. This phenomenon called RNA interference (RNAi) is considered as a defence mechanism against transposons integrated retroviral
genomes, or more generally viral infection. For
several years, RNAi could not be clearly observed
in somatic mammalian cells. A recent work have

shown that long double strand RNAs cannot

induce a visible degradation of the corresponding
mRNAs. This appears to be due to the fact that
the long double strand RNAs induce a potent
activation of a kinase (PKR) and of oligoadenylate synthetase. These mechanisms block mRNA
translation and trigger a non-specific degradation
of cellular mRNAs. This massive and non-specific
effect masks the RNAi. A transfection of preformed short double strand RNAs (shorter than
30 nucleotides), induced a specific RNAi without
the general mRNA degradation (Elbashir et al.,
2001).
Double strand RNAs ingested by Caenorhabditis elegans induce specific RNAi. Up to 5000
genes have been silenced by this extremely simple
and elegant method (Timmons and Fire, 1998). It
remains to determine if transgenesis may be implemented to synthesize long or short double
strand RNA to silence specific endogenous genes
in mammals.
Recent publications indicated that vectors expressing short double strand RNA under the action of polymerase III can specifically suppress
gene expression by an RNA interference effect
(Brummelkamp et al., 2002).
In practical term, it may happen than a given
gene construct contain information for the synthesis of a short double strand RNA which fortuitously inhibits the expression of a cellular gene
having a similar target sequence. Experimenters
should take this fact into consideration when they
design a gene construct to be transferred into cells
or living animals.

3.2.3. The inhibition of endogenous gene

expression by the o6erproduction of transdominant
negati6e proteins
The overproduction of a protein sharing only a
part of the properties of an endogenous protein
can anihilate the action of the latter. Indeed, the
former acts then as a decoy trapping the
molecules normally interacting with the normal
protein. Such an approach has met success several
times in transgenic animals. The major problem
to solve is generally to identify the mutant protein
capable of acting as a potent decoy without any
side-effect.

L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145160

All these methods have the theoretical advantage

of inhibiting in a specific, subtle, flexible and
reversible manner the expression of an endogenous
(or viral gene) by the only addition of a transgene.

3.3. The 6ectors for the conditional expression of

the transgenes
Specific gene promoters can define where and
when a transgene is expressed in animals. This
very popular approach has a fundamental limit:
the transgene is in these conditions at best strictly
dependent on the cellular machinery, which controls the promoter function. It is highly desirable
to induce and deinduce a transgene by a specific
inducer not acting on host genes. Several systems
have been proposed and are currently used. All of
them rely on the use of at least two genes. One
which may be under the control of a tissue specific
promoter codes for a transcription factor which
can specifically activate the gene of interest under
the action of a chemical inducer. Several inducers
have been defined: tetracycline and derivatives
(Rossi and Blau, 1998; Forster et al., 1999), rapamycin (Rivera et al., 1996), ecdysone (No et al.,
1996), RU 486 (Wang et al., 1997) and streptogramin (Fussenegger et al., 2000). All these
systems in their original version suffer from a
limitation. In the absence of the inducer, the gene
of interest is expressed at a background level
resulting from the action of the minimum promoter present in the construct. This background
may be considerably reduced by the action of a
silencer. In these systems, the silencer inhibit the
expression of the gene of interest in the absence of
the inducer. The presence of the inducer suppress
the effect of the silencer and allows the activator
to act (Rossi et al., 1998; Forster et al., 1999;
Freundlieb et al., 1999). These systems thus require the simultaneous contribution of three
genes, which must be introduced separately or not
into the experimental animals. They presently offer the best opportunity to evaluate in a subtle
manner the role of the genes.

3.4. The conditional homologous recombination

Specific DNA sequences have in nature the

153

capacity to recombine which high efficiency under

the action of a recombinase. Two of these sequences are currently used by experimenters. One
of them named LoxP can recombine under the
action of Cre recombinase from the bacteriphage
P1 (Nagy, 2000) The other named FRT from yeast
recombines under the action of the Flp recombinase (Umana et al., 2001). These DNA sequences
contain about 30 nucleotides, which are not found
in animal genomes. Several experimenters noticed
that high expression of the recombinases may be
cytotoxic. This has been attributed to a non-strict
recognition of genomic sequences by the enzymes
present in excess leading to an alternation of
genome DNA. These recombination systems are
being used to eliminate DNA fragments boardered by LoxP or FRT sequences when the corresponding recombinases are present (Kellendonk et
al., 1996; Utomo et al., 1999; Nagy, 2000). These
tools can also trigger specific recombination between chromosomes in which the LoxP or FRT
sequences have been previously introduced (Herault et al., 1998).
The Cre LoxP system can theoretically be used
to introduce foreign DNA sequences in a given
site of a genome. This implies first that a LoxP
sequence has been introduced in the genome by
gene addition or homologous recombination. A
gene construct containing the LoxP sequence may
recombine with the genomic LoxP in the presence
of the Cre recombinase and become integrated at
the site. This approach theoretically offers to experimenters the possibility to introduce various
gene constructs always in the same genomic site.
This reduces considerably the unpredictable position effect of chromatin on a transgene. The same
approach allows a foreign coding sequence to be
introduced in a given host gene and thus be in the
best location to be specifically expressed under the
control of the targeted gene (Wallace et al., 2000;
Gorman and Bullock, 2000; Day et al., 2000).
To be really exploitable this protocol must be
adapted. Indeed, for simple kinetic reasons, the
DNA fragment integrated into a LoxP site is
efficiently ejected by the Cre recombinase. To
enhance the chance of the foreign DNA sequence
to remain in the genomic LoxP site, two mutants
of LoxP can be used. The LoxP site resulting

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L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145160

from the recombination of the mutants bears both

mutations. This new LoxP sequence is no more
recognized by the recombinase. The integrated gene
is thus no more eliminate (Araki et al., 1997).
However, the mutated LoxP sequences have limited
capacity to recombine efficiently. This reduces the
interest of this method. Alternatively inverted LoxP
can be added in both the genomic and the foreign
DNA. This was shown to enhance considerably the
integration of the foreign DNA in the genomic
LoxP site (Feng et al., 1999; Schubeler et al., 2000).
An alternative recombination system to target
introduction of a foreign gene has been proposed
recently. This system named recombinase-mediated
cassette exchange (RMCE) is based on the use of
a single recombinase and of two FRT sequences.
One of the sequence is mutated and it cannot
recombine with the wild counterpart. The two FRT
sequences must be added in the genome at chosen
sites. The recombination vector is added in excess
with the Flp recombinase. The sequences of the
genome and the vector boardered by the FRT
sequences are exchanged with high efficiency. The
excess of the vector transferred to cells considerably
reduces the chance of a reciprocal recombination.
This exchange is thus a one way process. A combination of RMCE with a promoter trap vector
allows targeted recombination to occur in almost
100% of the cases (Baer and Bode, 2001).
In a study published several years ago, it was
reported that the specific expression of the Cre
recombinase gene in male germ line precursors
allows a high rate of recombination of LoxP
sequences in crossed mice harboring both genetic
modifications. The authors hypothesized that the
Cre recombinase expressed during spermatozoa
maturation was quite efficiently bound to the LoxP
sites allowing a recombination with a high frequency. This method named targeted meiotic recombination was used extensively to knock out
homeotic genes (Herault et al., 1998). It can potentially be used to efficiently target the introduction
of foreign genes in embryos.

4. Conclusions and perspectives

Since the first transgenic mice were born in 1980,

transgenesis techniques have been improved with a

clear recent acceleration. Among the most relevant
technical progress obtained in the past years several
are worth being mentioned.
The possibility to add and replace genes by
homologous recombination in cells further used to
generate transgenic animals by nuclear transfer into
enucleated oocytes is undoubtedly a major technical advance. The possibility to use the RNAi
phenomenon to knock genes out in a simplified
manner is essential to study gene function in
invertebrates and also possibly in mammals. The
discovery of insulators started helping experimenters to prepare gene constructs allowing a much
more predictable and reliable expression of the
transgenes. An increasing number of insulators will
be described in the coming years and this will offer
multiple possibilities to experimenters to optimize
their gene constructs. Presently insulators are generally defined as long genomic DNA sequences. In
a few cases only, the sequences carrying the insulating property have been found. Most libely, in future
relatively short sequences containing all the elements of insulators will be available allowing the
construction of compact and efficient expression
vectors.
The study and the use of insulators often requires
the manipulation of long DNA fragments. Several
new cloning vectors and particularly BAC are very
helpful for this purpose. It is essential that protocols have been defined to introduce foreign sequences into long DNA fragments by homologous
recombination in bacteria (Giraldo and Montoliu,
2001).
It is also worth noting that the use of the
Cre-LoxP and Flp-FRT systems has been improved
to induce in vivo recombination (Wunderlich et al.,
2001) or to introduce foreign DNA into targeted
sites of the genome (Feng et al., 1999; Schubeler et
al., 2000; Baer and Bode, 2001).
The improvement of the expression system based
on the control of transgene expression by tetracycline is also quite significant (Forster et al., 1999;
Freundlieb et al., 1999).
Transgenesis is and will remain limited by theoretical and technical problems. One limitation certainly comes from the fact that the gene transfer
into an animal is the come back of an isolated gene

L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145160

to the complexity of a whole living organism. It is

disappointing that almost 30% of gene knock-out
do not result in significant phenotypic modifications. The conditional gene knock-out and the
possibility to use RNAi to inhibit gene expression
will certainly reduce the proportion of failures in
this field of research.
Genes obviously contain multiple signals in their
transcription regulation regions but also in their
transcribed regions. Only part of these signals are
known. Gene constructions may result in the association of these signals in an inappropriate manner
leading to alternative splicing, mRNA destabilization etc. These kind of artefacts cannot be prevented as long as all the mechanisms of gene
expression are not better understood.
An increasing number of data indicate that
transgenes but also in some cases genes are expressed in a variegated manner. This mosaicism of
expression is considered to be due in number of
cases to the presence of retrotransposons in the
vicinity of the genes or transgenes. The retrotransposons are very abundant in mammalian genomes
and most of them are inactivated by DNA methylation. During embryo development, retrotransposons as genes are demethylated and thus activated
and later selectively remethylated. It appears more
and more that remethylation of transposons is
partly stockastic leading to an unpredictable activation or inactivation of their expression. It is admitted that transposons can stimulate or inhibit the
expression of the neighbor genes. Hence, genes and
transgenes may be subjected to the stock astic
activation of transposons. This phenomenon is
probably the most prominent epigenetic mechanism influencing gene and transgene expression
(Whitelaw and Martin, 2001; Symer and Bender,
2001; Rakyan et al., 2001). One of the function of
insulators seems to reduce to some extent the
impact of epigenesis on gene and transgene expression.
Improvements of transgenesis techniques are
expected. A better understanding of the phenomena which occur during cloning by nuclear transfer
should progressively contribute to enhance the
efficiency of transgenesis by this method.
Autonomously replicating vectors based on artificial chromosomes (Voet et al., 2001) or on

155

circular DNA structure (Kelleher et al., 1998)

would be of great help for transgenesis. Such
vectors are theoretically capable of giving an extremely high yield of transgenic animals. On the
other hand, the expression of transgenes is expected
to be high and reliable due to the fact that several
copies of the vectors may be present in each cell and
that the foreign DNA is not integrated and thus not
subjected to chromatin environment.
Gene mutation in situ or in test tube may perhaps
be improved by the use of ribo-deoxyribo-oligonucleotides (Graham et al., 2001) or of the bacterial
Rec A system (Pati, 1998).
The different tools to inhibit gene expression,
oligonucleotides forming a triple helix with DNA
or RNA, antisense RNA and ribozymes, are quite
attractive but the optimal conditions of their use
remain to be found.
The application of animal transgenesis for basic
research started in 1980 when the first transgenic
mice obtained by DNA microinjection. Indeed, the
first transgenes were not expressed although it was
perfectly active when transfected in cultured cells.
About one decade later this initial failure met an
explanation which led to the concept of LCR
insulator and chromatin opener. This pioneer experiment revealed that some essential mechanisms
of gene expression can be studied only in vivo.
More generally, it is clear that the availability of
the majority of human and laboratory animal genes
identified by systematic genome sequencing or
systematic chemical mutation in mouse (Brown and
Balling, 2001) will require the frequent implementation of transgenesis to study their function. Gene
addition, gene replacement, gene mutation possibly
by the use of RDO or of the Rec A bacterial system
and gene trap (Cecconi and Meyer, 2000; Medico
et al., 2001) will be more and more used for that
purpose.
It is admitted that up to 5000 genes have been
knocked out by homologous recombination in
mouse. These experiments include the study of gene
function but also the generation of models for the
study of human diseases. Mouse is presently the
most utilized species for this purpose. It is now
more and more evident that Drosophila and human
share many of their genes and interestingly more
than 60% of the genes implicated in human diseases

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L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145160

have Drosphila orthologues (Bernards and Hariharan, 2001). Gene manipulation is easier in
Drosophila than in mouse. It is therefore expected
that Drosphila will be more systematically used
for the study of human diseases. The same reasoning can be applied to another invertebrate,
Caenorhabditis elegans, the genome of which has
also recently been fully sequenced. Other species,
namely rat (Charreau et al., 1996), rabbit (Fan et
al., 1999) and pig will be more frequently used in
the future to study human diseases. Several arguments converge in this direction. These mammalian species have in some cases metabolic
properties closer to human than mouse and
surgery is also more easily performed on larger
animals. The fact that gene replacement by homologous recombination will probably become a
reality in the coming years in theses species via the
cloning technique is an additional argument.
The vectors to induce gene mutation by homologous recombination are being improved and
they tend to mimic human diseases in a more
subtle manner (Petters and Sommer, 2000). A case
in point is in the field cancer. A recent work
showed that some vectors allow the k-ras gene to
be mutated in a sporadic manner and in single
cells in the adult animal. This elegant approach
reflects more precisely the natural genetic alterations which lead to the development of a tumor
(Berns, 2001; Johnson et al., 2001).
After the birth of the first transgenic mice expressing their transgene, the rat human growth
hromone gene, it was suggested that transgenic
animals could become an industrial source of
recombinant proteins for pharmaceutical use.
This hypothesis is close to become a reality
(Houdebine, 2000).
The increasing shortage of organs and cells for
transplantation to humans inclines to use these
biological materials from pig. This can become a
reality only when the rejection mechanisms will be
understood and controlled and when expression
of endogenous retrovirus genomes will be inhibited. Transgenic animals play a central role in this
medical project (Houdebine and Weill, 1999).
The use of transgenesis to improve animal
breeding has received little attention so far. This is
a glaring contrast with what happens with geneti-

cally modified plants already available for animal

and human consumption. This is obviously due to
the technical hurdles and the cost of transgenesis
in farm animals. The possibility to use cloning
technique for gene addition and replacement in
farm animals has opened new avenues for
breeding.
Among the projects in development, the following are worth being mentioned.
Pigs and sheep expressing growth hormone or
IGF1 genes have an amplified muscle development with no alteration of their health. Several
fish species have significant or considerable
growth acceleration when they express a growth
hormone transgene. Their use in aquaculture
awaits for the design of breeding conditions preventing any dissemination of the transgenesis in
wild water. Indeed, a study using medaka as a
model revealed that the GH transgene might have
deleterious and long term effects on wild fish
(Muir and Howard, 1999).
Transgenic mice expressing the lysostaphin gene
in their mammary gland have become resistant to
infection by Staphylococcus aureus (Kerr et al.,
2001). Transgenic farm animals could be protected from mastitis by the same transgene. Number of animal diseases are expected to be
prevented by the action of various transgenes
(Mu ller, 2001).
Transgenic mice expressing a transdominant
negative myostatin gene show an increased muscle
development after birth mimicking the culard
trait. This transgene might enhance meat production in farm animals (Yang et al., 2001).
Transgenic mice and pigs expressing a phytase
gene in the salivary gland (Ward, 2001; Golovan
et al., 2001a,b) or intestine have become capable
of digesting phytic acid present in plants. This
leads to a quite significant reduction of phosphate
release in environment and to accelerated growth
of the animals.
The transfer of genes involved in wool synthesis
into sheep has modified fiber composition. The
aim of the study is to enhance wool growth but
mainly to improve wool properties (Bawden et al.,
1999).
Gene addition and replacement are potent tools
to optimize milk composition for animal and human consumption (Houdebine, 1998). The secre-

L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145160

tion of a lactase in the milk of transgenic mice

reduced lactose concentration (Jost et al., 1999).
The transfer of a gene coding for a mutated
human phenylalanine-free a-lactalbumin into cow
allows the production in milk of this protein for
patients suffering from phenylketoneurea (PPL
Therapeutics, unpublished data).
Many other possibilities can be envisaged in
this field (Bulfied, 2000; Ward, 2000). Transgenesis, including the intentional dissemination of the
transgenes in herds, will remain a heavy and
costly task in farm animals. A success thus implies
that relevant genes have been identified to expect
a beneficial impact for breeders or consumers.
The systematic study of farm animal genomes
which has been undertaken several years ago offers this possibility.

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