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 D� eloppement 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
biologists. The first gene transfer into mouse using
1. Introduction
isolated DNA revealed that the generation of
animals stably harboring foreign DNA (Gordon
The discovery of genetic code about 40 years
et al., 1980) and having modified phenotypic
ago suggested that gene isolation and transfer into
properties (Palmiter et al., 1982) was possible.
living organisms would become major tools for
These pioneer and fascinating experiments also
revealed some of the limits of transgenesis: the
* Tel.: [phone]; fax: [phone].
generation of transgenic animals by gene microin-
E-mail address: [e-mail] (L.-M.
Houdebine). jection appeared laborious; the first transgene un-
0168-1656/02/$ - see front matter � 2002 Elsevier Science B.V. All rights reserved.
PII: S0168-1656(02)00129-3
146 L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145 160
expectedly remained very poorly active or inac- gene can be transferred using different methods
tive; the growth hormone gene induced an over- according to animal species.
growing of the transgenic mice with numerous
physiological side-effects. These observations re- 2.1. DNA microinjection
vealed that the reintegration of an isolated gene
into the genome of an animal may generate com- The direct DNA microinjection into the pronu-
plex and unpredictable biological situations. clei of embryos was the first technique which led
Direct gene injections were extended to three to regular and relatively easy success in mammals.
others mammals in 1985 (Hammer et al., 1985) Essentially the same protocol is followed in
and later to several lower vertebrates and inverte- mouse, rat, rabbit, pig, sheep, goat and cow,
brates. Gene replacement by homologous recom- however with a decreasing yield of transgenic
bination was achieved in 1989 (Capecchi, 1989). animals from mouse to cow. Bovine has a slow
As many as 5000 mouse genes had been inacti- reproduction rate. The number of embryos gener-
vated by this method in 2000. ated by superovulation is low and the success of
The better knowledge of gene structure and microinjection appeared accessible only if em-
function allows the preparation of recombinant bryos were prepared in vitro after oocyte matura-
genes having a more predictable expression in tion and fertilization followed by in vitro
transgenic animals. development of the microinjected to the blastocyst
It is now well-established that transgenesis is stage (Krimpenfort et al., 1991). This method
one of the major tools of biologists to study gene remains laborious and costly.
expression and function. Transgenesis is still go- In lower vertebrates and invertebrates, pronu-
ing to be used more extensively and systematically clei are not visible gene. Microinjection must
with the identification of all the human genes. therefore be performed in cytoplasm, using much
Transgenesis also plays an essential role for appli- larger amounts of DNA. For unknown reasons,
cations in the medical and agronomic fields. The the success of this approach is quite variable from
study of human diseases is greatly facilitated by one species to another. It proved efficient in sev-
the generation of transgenic animals mimicking eral fish species and mainly in salmonids (Devlin,
health disorders or allowing the evaluation of new 1997). It remains inefficient in the laboratory fish
pharmaceuticals. Transgenic pigs are expected to medaka as well as in xenopus and chicken. In
be the source of organs and cells for transplanta- these species, foreign DNA usually does not inte-
tion to humans. Recombinant proteins of phar- grate into the genome of the animals.
maceuticals interest are being prepared in the milk In insects (Drosophila) and worms (Caenorhab-
of transgenic animals. A few lines or farm animals ditis elegans) (Thierry-Mieg et al., 1997), foreign
having improved breeding properties have been DNA is injected into gonad syncytium.
generated and are under study for human
consumption. 2.2. The use of transposons
Despite this impressive and growing success,
transgenesis still suffers from many imperfections. In several species, foreign DNA injected in
The present paper aims at describing the state of embryo cytoplasm becomes very rarely integrated
the art in the animal transgenesis field. in the genome. This is the case for medaka,
Drosophila, chicken, silk worms etc. In order to
enhance the frequency of integration, several tools
2. The methods to generate transgenic animals have been implemented. One possibility consists
in generating breaks in host DNA by injecting
The establishment of stable transgenic animals low amounts of restriction enzymes. The DNA
implies that the foreign DNA is present in repair mechanism restores DNA and integrates
gametes or one-cell embryos to allow its transmis- the foreign DNA. This approach proved to be
sion to progeny. To reach this goal, the foreign uneasy to manage. Low concentrations of restric-
L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145 160 147
tion enzyme, have no significant effects whereas genome. This is not the case for the piggy-Bac
high concentrations are deleterious for the host transposon, which has a narrow spectrum of hosts
genome. The optimum protocol was not found and proved to be fully stable over 15 generations
and this approach has not been retained. in transgenic silk worms.
Another possibility consists of using vectors
which have the intrinsic capacity to integrate in a 2.3. DNA transfer into gametes
genome with high efficiency. Retrotransposons
and retrovirus belong to this category. One logical approach to generate transgenic
Transposons are DNA sequences which contain animals may theoretically consist of introducing
at least one gene coding for a transposase and foreign DNA in gametes before fertilization.
motives located on both ends of the transposons The incubation of spermatozoa in the presence
the role of which is to trigger integration. Trans- of DNA followed by in vivo or in vitro fertiliza-
posons sequences are transcribed in RNA, which tion led to the generation of transgenic mice, fish,
drive transposase synthesis. The RNA is retro- chicken, rabbits, pigs, sheep and cows. However,
transcribed in DNA, which integrates in the mul- the results obtained with this method are inconsis-
tiple sites of the genome under the action of the tent. The yield of transgenic animals is usually
transposase. Numerous families of transposons low and largely unpredictable. Moreover, the inte-
have been identified. grated DNA is most of the times profoundly
To become a vector for gene transfer, a trans- rearranged and no more functional. This phenom-
poson must be genetically modified. The trans- ena can seemingly be greatly attenuated by select-
posase gene must be deleted to make space for a ing the most appropriate ejaculates and by
foreign gene and to prevent the recombinant removing DNAse by repeated washing and addi-
transposon to disseminate in an uncontrolled tion of DNAse inhibitors (Baccetti and
manner. This recombinant vector is unable alone Spadafora, 2000). This method in its improved
to integrate into a genome. The exogenous trans- version might become attractive in future for
posase must complement the vector. In practice, a some species.
circular plasmid containing a construct capable of In xenopus, gene transfer by spermatozoa was
expressing the transposase gene is injected with achieved successfully but on condition to alter
the recombinant vector. This allows the integra- sperm membrane by an incubation with detergent
tion of the foreign gene with the vector whereas or by freezing and thawing to allow foreign DNA
the assistant plasmid is rapidly degraded. to penetrate into the cell. After this first step,
Several transposons have been used success- spermatozoa have become inactive and intracellu-
fully. Transposon P have used for years to gener- lar sperm injection must be performed to obtain
ate transgenic Drosophila (Kayser, 1997). The fertilization (Marsh-Armstrong et al., 1999). This
mariner transposon originally found in method was applied successfully in mouse but it
Drosophila and adapted to different species is appeared no more attractive than the conven-
efficient to transfer gene in medaka (Hackett et tional microinjection (Perry et al., 1999). This
al., 2002), chicken (Shermann et al., 1998) and elegant protocol is the only available to generate
mouse (Dupuy et al., 2002). transgenic xenopus. It might be used for other
A recent work showed that transgenic silk species for which microinjection is inefficient.
worm expressing the GFP gene could be obtained A quite attractive tool has been recently pro-
using the piggy-Bac transposon (Tamura et al., posed. Linear DNA containing a functional gene
1999). was bound in a non-covalent manner to a mono-
Transposons are potent and flexible tools which clonal antibody, which recognizes a spermatozoa
might be used for gene therapy. They also appear specific antigen. The spermatozoa were incubated
safe although the mariner transposon seems to be with the antibody-DNA complex and further used
complemented to some extent by endogenous to fertilize oocyte by in vitro fertilization in
transposases and spread at a low rate in the host mouse, by artificial insemination in chicken and
148 L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145 160
by injection into uterine horn in pig. In all become integrated. This method is laborious and
cases, up to 30% of born animals were transgenic. less attractive than cloning to generate transgenic
The transgenes were expressed and transmitted cows. It is the only technique, which allowed the
to progency without any rearrangement. Interest- generation of transgenic primates so far.
ingly the same antibody recognized the same anti-
gen in several mammals including human and in 2.4. The use of retro iral ectors
lower vertebrates. This method might greatly sim-
plify gene addition in animals (Qian et al., Retroviral vectors are under intensive study
2001a,b). to tentatively transfer gene to human somatic
Experiments aiming at transferring foreign gene cells and proceed to gene therapy. Similar vectors
into sperm precursors either in vivo or in vitro have been designed to transfer foreign genes
are in course (for review articles see Baccetti and into mammalian embryonic cells. This approach
Spadafora, 2000). Although encouraging, the re- proved to be laborious and less efficient than
sults obtained by different groups indicate that classical microinjection. A family of vectors
additional studies are required before this ap- capable of infecting chicken primordial germ cells
proach may become an attractive alternative to and of generating transgenic animals has
the other methods. been described (Ronfort et al., 1997). Although
The mechanism of gene transfer into epididy- laborious this method remains the only, which
mal spermatozoa by injection of a DNA-transfec- allowed repeatedly to transfer foreign gene into
tant complex into testis is under study. This chicken.
method permitted the generation of limited num- A recent unpublished work carried out in D.
ber of transgenic animals so far (Sato et al., Baltimore laboratory has shown that lentiviral
2002). A retroviral vector carrying the -galac- vectors transfer foreign gene with quite high effi-
tosidase gene was used to infect male germ-line ciency in one cell embryo. This approach appears
stem cells. These cells was further transplanted much simpler than microinjection and might be-
into the testis of unfertile recipient mice. Approxi- come used extensively in future (Lois et al., 2002).
mately 4.5% of progeny from these males were
transgenic and the transgene was transmitted to 2.5. Gene transfer using embryonic cells
progeny and expressed in subsequent generations
(Nagano et al., 2001). Gene replacement by homologous recombina-
In case, sperm precursors could be cultured and tion is performed in routine in bacteria and yeast.
matured, they could be used not only to add It can be achieved in somatic mammalian cells
foreign genes but also to replace host genes by although with a relatively poor efficiency. For
homologous recombination. unknown reasons, homologous recombination is
DNA microinjection into oocyte was never or more frequent in pluripotent embryonic cells.
very rarely followed by the birth of transgenic This approach is very attractive since it can lead
animals. Recent experiments showed that retrovi- to specific gene inactivation, to targeted point
ral vectors introduced between the zona pellucida mutation in an animal genome or to the replace-
and the plasma membrane of the oocytes can be ment of a given gene by a non-related one.
implemented to generate transgenic cows (Chan Homologous recombination is a rare event.
et al., 1998) and monkeys (Chan et al., 2001). Cells in which it occurred must be selected and
This success was met with viral particles contain- further used to generate a living embryo. This
ing the VSV-G envelope, which is known to rec- proved to be feasible on condition to use embry-
ognize membrane phospholipids of all cell types. onic stem cells capable of forming chimeric em-
The infection was performed at a period when the bryos after microinjection into blastocysts or
nuclear membrane of the oocyte was non-existent. morula (Viville, 1997). Although laborious this
This greatly enhanced the chance of the recombi- protocol has become popular and genes are fre-
nant retroviral to reach the host genome and quently inactivated in mouse.
L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145 160 149
Despite repeated efforts, the extension of this from early embryos and not previously cultured.
method to species other than mouse failed. This is These observations gave credence to the idea that
clearly due to the fact that the recombined ES animal cloning by nuclear transfer was possible
cells have more or less the capacity to participate only with nuclei from totipotent or pluripotent
to the development of chimeric embryos but that cells.
transmission of the mutation to progeny has been A systematic study carried out in sheep revealed
observed so far only in two mouse lines and that fully differentiated cells as well as embryonic
essentially of the 129/SV line (Smithies, 2001). For or foetal cells could successfully give their genes
years, it was admitted that mouse was the model to generate normal animals. (Wilmut et al., 1997).
for the use of ES cells to mutate genes in a The conditions defined for cloning animals start-
targeted manner. The systematic lack of success ing from differentiated cells was retained soon
met in rat, rabbit, chicken, pig, sheep and cow after to generate transgenic sheep by gene addi-
now inclines to consider that the so-called ES cells tion (Schnieke et al., 1997). Gene replacement was
cannot be used for the germinal transmission of a also achieved in sheep (Ayares, 1999; McCreath et
mutation except in two mouse lines systematic al., 2000) and pig (Dai et al., 2002; Lai et al.,
studies to tentatively identify genes involved in the 2002). This experiment is not an easy task. Several
two mouse lines are in course. They might con- independent problems must be solved to meet the
tribute to define conditions to use ES cells and success. Homologous recombination is less fre-
chimeric animals to replace genes in various quent in differentiated cells than in ES cells. In
species. the experiments described above, fS
tal fibroblasts
It should be mentioned that in a work pub- were used to add genes by transfection and re-
lished recently, it was reported that appropriate place gene by homologous recombination. These
vectors are capable of inducing targeted gene cells, although of fS
tal origin, have a limited
transfer into Drosphila by homologous recombi- number of possible multiplication cycles which
nation (Bernards and Hariharan, 2001). corresponds roughly to the number of passages
Two recent studies indicate that chicken cul- required to select clones in which homologous
tured primordial germ cells retransferred to em- recombination took place. The culture of the re-
bryos can participate to their development and combined cells must therefore be performed with
transmit their genes to progeny (Petitte et al., a particular care. On the other hand, it has been
2002). ES like cells from medaka embryo capable repeatedly observed that cells from adults and
of generating chimaeric animals have also been cells cultured for long periods of time is a less
described and are potential tools for gene transfer efficient material for cloning by nuclear transfer.
and targeting in fish (Collodi, 2001). The reasons why these phenomena occur are un-
known. Mutation of essential genes for embryo
2.6. Gene transfer by nuclear transfer development may occur progressively in living
animals and in cultured cells. Alternatively, chro-
In front of the repeated failure met with ho- matin may progressively adopt a less reversible
mologous recombination in ES cells followed by conformation which reduces the chance of a suc-
the formation of chimeric embryos, experimenters cessful cloning. Interestingly, a recent study
addressed the problem by a quite different showed that the donor genome in bovine embryos
method. generated by nuclear transfer from somatic cells is
Experiments carried out about 40 years ago aberrantly methylated (Kang et al., 2001). This
revealed that the nucleus from embryonic cells may have inactivated genes required for embryo
experimentally transferred into xenopus enucle- development.
ated oocytes gave birth to living animals. This In a recent publication, it was reported that
method was extended to sheep about 15 years ago cloned sheep having knocked-out PrP gene did
but the success was restricted to experiments in not survive after birth (Wells, 2001; Denning et
which cells used as nuclear donors were taken al., 2001). This illustrates the difficulty of the task.
150 L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145 160
The cloning approach to knock gene out was nomic enhancers and their extinction by silencers
used successfully in mouse with no evidence that seem not symmetrical. The second is a phe-
it is less laborious than the classical ES cell- nomenon more frequent and more potent than
chimera method (Readeout et al., 2000).
the first. Numerous observations indicate that a
Gene addition by the cloning technique have
transgene is generally poorly expressed (i) when it
been extended to goat (Reggio et al., 2001) and
contains a cDNA rather than the corresponding
cow (Arat et al., 2001; Zakhartchenko et al.,
genomic DNA sequence with its introns (ii) when
2001) and pig (Dai et al., 2002). Three groups
multiple copies are integrated in the same site (iii)
obtained cloned pigs independently and by differ-
when a bacterial gene is used. This is reminiscent
ent methods. Gene knock-out was recently
of the extinction of transposons and retroviral
achieved in the pig. Moreover, cloning was suc-
genomes. The mechanisms involved in these cases
cessful in the rabbit (Chesn� et al., 2002).
might also operate to silence transgenes.
This suggests that gene replacement will be-
Several approaches are possible to reduce ec-
come a reality in several species other than
topic expression and silencing of transgenes.
mouse in the coming years. Obviously, quite sig-
In eucaryotic genomes, genes and gene clusters
nificant progress in the cloning technique and
are boardered by DNA regions capable of pre-
mainly in the culture of transfected cells to be
venting interactions between neighbor genes.
used as nuclear donors must be done before reg-
These sequences which have not yet been well
ular success can be hoped. Gene addition by the
described are named insulators (Bell et al., 2001).
cloning method is preferred by experimenters
Some of insulators have a potent silencer ef-
working in ruminants rather than the classical
fect. This is the case for the 5 HS4 region from
microinjection technique. It is by no means cer-
the locus control region (LCR) of the chicken
tain that cloning will be adopted to add gene in
-globin locus. This sequence added in duplicate
the highly proliferic species (mouse, rat, rabbit
to transgenes strongly attenuated position effect
and pig). Microinjection remains presently more
(Taboit-Dameron et al., 1999).
advantageous.
Numerous data support the idea that insula-
tors stimulate transgene expression by preventing
the local formation of inactive heterochromatin.
3. The methods to control gene expression
However, it is not presently clear if insulators
taken as a whole have just a silencer effect or if
3.1. The reliability of transgene expression
some of them contain enhancers and must be
considered as chromatin openers.
Many transgenes work poorly. Their expres-
Most likely, an increasing number of insulators
sion often is very low or not specific of the
are going to be described and used in the coming
promoter added in the gene construct. It is gener-
years. An alternative to the use of identified insu-
ally admitted that these events result from posi-
lators may consist of utilizing long genomic
tion effects in chromatin. Enhancers and silencers
DNA fragments ( 100 kb) surrounding the gene
from neighbor genes are supposed to activate or
of interest. This genomic material is generally
inactivate transgenes. Precise experimental data
quite active as transgene. One of the difficulty of
support this view. Genomic DNA surrounding a
this approach is that long DNA fragment must
well-expressed transgene favors the expression of
be recombined to introduce foreign DNA. Vari-
another transgene when added to it. The phe-
ous methods based or homologous recombina-
nomena which govern this effect seem however
tion in yeast or preferably in bacteria have been
rather complex. Subtle interactions between the
transgene proper and the genomic DNA se- defined to prepare gene constructs with long
quences seems to influence the expression of the DNA fragments (Giraldo and Montoliu, 2001).
foreign gene (Cranston et al., 2001). In addition to the use of insulators, a certain
The stimulation of transgene expression by ge- number of rules must be respected to avoid low
L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145 160 151
expression of transgenes. When cDNAs rather early stage of embryo development. This does not
than genes are to be used, the addition of a least reflect the subtle regulation to which genes are
one intron before the cDNA is required. The normally submitted. Conditional recombination
gene construct must contain as little as possible
may be triggered in a given cell type of the
of CpG motives specially in the promoter region.
animal and at a given period of its life. This
Unexpectedly, house-keeping genes which have
implies that two LoxP sequences from P1 phage
CpG stretches are generally expressed in all cell
are initially added to both ends of the targeted
types with limited regulation. The same genes are
gene.
frequently extinguished when they have become
LoxP sequences can recombine with high effi-
transgenes (Cohen-Tannoudji et al., 2000). A
ciency and specificity under the action of the Cre
chemical synthesis of the cDNA aiming at reduc-
recombinase from P1 phage. This recombination
ing the number of CpG and at choosing the most
eliminates the genomic sequence located between
commonly used codons in animal cells may be
the two LoxP and knock the gene out. The pres-
recommended, specially for bacterial genes
ence of Cre recombinase induces the LoxP re-
(Henry et al., 1999). Cryptic splicing sites are
combination. The enzyme can be synthesized
sometimes introduced in gene constructs. This
from plasmids transferred to cells and transiently
may lead to the synthesis of truncated and non-
expressed. Alternatively, adenoviral vectors har-
functional mRNAs. An examination of the vector
boring a construct expressing Cre recombinase
sequence on a case by case basis must be done to
gene may be injected into a given tissue to induce
avoid these problems. These recommendations
local LoxP recombination. Mice harboring a sta-
and others have been detailed elsewhere
ble transgene containing the Cre recombinase
(Houdebine et al., 2002).
gene under the control of a tissue specific pro-
In a certain number of situations, it may be
moter may be crossed with other transgenic mice
advantageous to express two cistrons from the
in which targeted introduction of LoxP sequences
same construct. Two independent transcriptional
has been performed. The inactivation of the gene
units may be cloned in the same vector. Alterna-
induced by LoxP recombination will occur spe-
tively more compact vectors may be prepared
cifically in the tissue in which the promoter is
using internal ribosome entry site (IRES). These
active. Another level of control can be added to
elements are known to allow the translation of
the system. Fusion genes containing the Cre re-
two adjacent cistrons when added between these
combinase and the tamoxifen or RU 486 receptor
coding sequences. Numerous studies have shown
sequences have been generated (Wunderlich et al.,
that the action of IRES is somewhat unpre-
2001) The Cre recombinase activity of these fu-
dictable. A systematic study revealed that, for
sion proteins is induced by the administration of
some IRES at least, the terminator codon of the
tamoxifen or RU 486 respectively. These systems
first cistron must be at about 80 nucleotides of
offer an additional control of LoxP recombina-
the IRES to allow an optimal expressing of the
tion and of gene knock out. The systems for the
second cistron (and even of the first) (Houdebine
control of transgene expression based on the use
and Attal, 1999).
of inducers such tetracycline described below can
also be implemented to tune Cre recombinase
3.2. The ectors to inhibit endogenous gene
gene expression (Nagy, 2000).
expression
A certain number of mouse lines express the
Cre recombinase gene in specific tissues. They can
Homologous recombination is an excellent way
be obtained from the laboratories in which they
to suppress the expression of a gene in a whole
were prepared or from the Jackson Laboratory
animal. This method suffers from several intrinsic
limitations. It is a laborious method not efficient or Charles River Company.
in all cases. On the other hand, the method im- Alternative approaches may be envisaged in
plies that the targeted gene is knocked out at the some cases.
152 L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145 160
3.2.1. The inhibition of endogenous gene shown that long double strand RNAs cannot
expression by the formation of triple helix induce a visible degradation of the corresponding
A DNA or RNA region rich in pyrimidine may mRNAs. This appears to be due to the fact that
form a stable triple helix with a complementary the long double strand RNAs induce a potent
purine rich DNA or RNA strand. The triple helix activation of a kinase (PKR) and of oligoadeny-
structure specifically prevents transcription and late synthetase. These mechanisms block mRNA
translation (Upegui-Gonzalez et al., 2000). Al- translation and trigger a non-specific degradation
though attractive, these methods have never been of cellular mRNAs. This massive and non-specific
proved efficient in transgenic animals. Additional effect masks the RNAi. A transfection of pre-
studies are required to evaluate to which degree formed short double strand RNAs (shorter than
they may really be reliable tools to control expres- 30 nucleotides), induced a specific RNAi without
sion of endogenous genes. the general mRNA degradation (Elbashir et al.,
2001).
3.2.2. The inhibition of endogenous expression by Double strand RNAs ingested by Caenorhabdi-
antisense RNAs tis elegans induce specific RNAi. Up to 5000
Natural mechanisms capable of blocking selec- genes have been silenced by this extremely simple
tively the expression of endogenous genes are and elegant method (Timmons and Fire, 1998). It
based on the expression of antisense RNAs which remains to determine if transgenesis may be im-
form hybrids with the targeted mRNAs and pre- plemented to synthesize long or short double
vent their translation. Artificial antisense RNAs strand RNA to silence specific endogenous genes
are being used but most of the times with a low in mammals.
efficiency. It is admitted that this is essentially due Recent publications indicated that vectors ex-
to the fact that the antisense RNAs and the pressing short double strand RNA under the ac-
targeted mRNA sequences have little chance to tion of polymerase III can specifically suppress
interact. Both partners have often secondary gene expression by an RNA interference effect
structure and they are associated with proteins (Brummelkamp et al., 2002).
rendering their contact unlikely. In practical term, it may happen than a given
The same is true for the antisense RNAs having gene construct contain information for the synthe-
ribozyme activity capable of cleaving the targeted sis of a short double strand RNA which fortu-
mRNAs at specific sites. A systematic search of itously inhibits the expression of a cellular gene
the accessible sites in targeted mRNAs may be having a similar target sequence. Experimenters
done by different methods. On the other hand, the should take this fact into consideration when they
ribozymes may be inserted in one of the loops of design a gene construct to be transferred into cells
a tRNA or other small RNAs. A recent study or living animals.
showed that when the ribozyme is associated with
an RNA helicase, any site of an RNA can be 3.2.3. The inhibition of endogenous gene
reached and cleaved by the ribozyme (Warashina expression by the o erproduction of transdominant
et al., 2001). negati e proteins
Numerous experiments have shown that in The overproduction of a protein sharing only a
plants, in invertebrates and in mammal embryos, part of the properties of an endogenous protein
double strand RNAs strongly induce a specific can anihilate the action of the latter. Indeed, the
degradation of the mRNA having the same RNA former acts then as a decoy trapping the
sequence. This phenomenon called RNA interfer- molecules normally interacting with the normal
ence (RNAi) is considered as a defence mecha- protein. Such an approach has met success several
nism against transposons integrated retroviral times in transgenic animals. The major problem
genomes, or more generally viral infection. For to solve is generally to identify the mutant protein
several years, RNAi could not be clearly observed capable of acting as a potent decoy without any
in somatic mammalian cells. A recent work have side-effect.
L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145 160 153
All these methods have the theoretical advantage capacity to recombine which high efficiency under
of inhibiting in a specific, subtle, flexible and the action of a recombinase. Two of these se-
reversible manner the expression of an endogenous quences are currently used by experimenters. One
(or viral gene) by the only addition of a transgene. of them named LoxP can recombine under the
action of Cre recombinase from the bacteriphage
3.3. The ectors for the conditional expression of P1 (Nagy, 2000) The other named FRT from yeast
the transgenes recombines under the action of the Flp recombi-
nase (Umana et al., 2001). These DNA sequences
Specific gene promoters can define where and contain about 30 nucleotides, which are not found
when a transgene is expressed in animals. This in animal genomes. Several experimenters noticed
very popular approach has a fundamental limit: that high expression of the recombinases may be
the transgene is in these conditions at best strictly cytotoxic. This has been attributed to a non-strict
dependent on the cellular machinery, which con- recognition of genomic sequences by the enzymes
trols the promoter function. It is highly desirable present in excess leading to an alternation of
to induce and deinduce a transgene by a specific genome DNA. These recombination systems are
inducer not acting on host genes. Several systems being used to eliminate DNA fragments board-
have been proposed and are currently used. All of ered by LoxP or FRT sequences when the corre-
them rely on the use of at least two genes. One sponding recombinases are present (Kellendonk et
which may be under the control of a tissue specific al., 1996; Utomo et al., 1999; Nagy, 2000). These
promoter codes for a transcription factor which tools can also trigger specific recombination be-
can specifically activate the gene of interest under tween chromosomes in which the LoxP or FRT
the action of a chemical inducer. Several inducers sequences have been previously introduced (Her-
have been defined: tetracycline and derivatives ault et al., 1998).
(Rossi and Blau, 1998; Forster et al., 1999), ra- The Cre LoxP system can theoretically be used
pamycin (Rivera et al., 1996), ecdysone (No et al., to introduce foreign DNA sequences in a given
1996), RU 486 (Wang et al., 1997) and strep- site of a genome. This implies first that a LoxP
togramin (Fussenegger et al., 2000). All these sequence has been introduced in the genome by
systems in their original version suffer from a gene addition or homologous recombination. A
limitation. In the absence of the inducer, the gene gene construct containing the LoxP sequence may
of interest is expressed at a background level recombine with the genomic LoxP in the presence
resulting from the action of the minimum pro- of the Cre recombinase and become integrated at
moter present in the construct. This background the site. This approach theoretically offers to ex-
may be considerably reduced by the action of a perimenters the possibility to introduce various
silencer. In these systems, the silencer inhibit the gene constructs always in the same genomic site.
expression of the gene of interest in the absence of This reduces considerably the unpredictable posi-
the inducer. The presence of the inducer suppress tion effect of chromatin on a transgene. The same
the effect of the silencer and allows the activator approach allows a foreign coding sequence to be
to act (Rossi et al., 1998; Forster et al., 1999; introduced in a given host gene and thus be in the
Freundlieb et al., 1999). These systems thus re- best location to be specifically expressed under the
quire the simultaneous contribution of three control of the targeted gene (Wallace et al., 2000;
genes, which must be introduced separately or not Gorman and Bullock, 2000; Day et al., 2000).
into the experimental animals. They presently of- To be really exploitable this protocol must be
fer the best opportunity to evaluate in a subtle adapted. Indeed, for simple kinetic reasons, the
manner the role of the genes. DNA fragment integrated into a LoxP site is
efficiently ejected by the Cre recombinase. To
3.4. The conditional homologous recombination enhance the chance of the foreign DNA sequence
to remain in the genomic LoxP site, two mutants
Specific DNA sequences have in nature the of LoxP can be used. The LoxP site resulting
154 L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145 160
from the recombination of the mutants bears both transgenesis techniques have been improved with a
mutations. This new LoxP sequence is no more clear recent acceleration. Among the most relevant
recognized by the recombinase. The integrated gene technical progress obtained in the past years several
is thus no more eliminate (Araki et al., 1997). are worth being mentioned.
However, the mutated LoxP sequences have limited The possibility to add and replace genes by
capacity to recombine efficiently. This reduces the homologous recombination in cells further used to
interest of this method. Alternatively inverted LoxP generate transgenic animals by nuclear transfer into
can be added in both the genomic and the foreign enucleated oocytes is undoubtedly a major techni-
DNA. This was shown to enhance considerably the cal advance. The possibility to use the RNAi
integration of the foreign DNA in the genomic phenomenon to knock genes out in a simplified
LoxP site (Feng et al., 1999; Schubeler et al., 2000). manner is essential to study gene function in
An alternative recombination system to target invertebrates and also possibly in mammals. The
introduction of a foreign gene has been proposed discovery of insulators started helping experi-
recently. This system named recombinase-mediated menters to prepare gene constructs allowing a much
cassette exchange (RMCE) is based on the use of more predictable and reliable expression of the
a single recombinase and of two FRT sequences. transgenes. An increasing number of insulators will
One of the sequence is mutated and it cannot be described in the coming years and this will offer
recombine with the wild counterpart. The two FRT multiple possibilities to experimenters to optimize
sequences must be added in the genome at chosen their gene constructs. Presently insulators are gen-
sites. The recombination vector is added in excess erally defined as long genomic DNA sequences. In
with the Flp recombinase. The sequences of the a few cases only, the sequences carrying the insulat-
genome and the vector boardered by the FRT ing property have been found. Most libely, in future
sequences are exchanged with high efficiency. The relatively short sequences containing all the ele-
excess of the vector transferred to cells considerably ments of insulators will be available allowing the
reduces the chance of a reciprocal recombination. construction of compact and efficient expression
This exchange is thus a one way process. A combi- vectors.
nation of RMCE with a promoter trap vector The study and the use of insulators often requires
allows targeted recombination to occur in almost the manipulation of long DNA fragments. Several
100% of the cases (Baer and Bode, 2001). new cloning vectors and particularly BAC are very
In a study published several years ago, it was helpful for this purpose. It is essential that proto-
reported that the specific expression of the Cre cols have been defined to introduce foreign se-
recombinase gene in male germ line precursors quences into long DNA fragments by homologous
allows a high rate of recombination of LoxP recombination in bacteria (Giraldo and Montoliu,
sequences in crossed mice harboring both genetic 2001).
modifications. The authors hypothesized that the It is also worth noting that the use of the
Cre recombinase expressed during spermatozoa Cre-LoxP and Flp-FRT systems has been improved
maturation was quite efficiently bound to the LoxP to induce in vivo recombination (Wunderlich et al.,
sites allowing a recombination with a high fre- 2001) or to introduce foreign DNA into targeted
quency. This method named targeted meiotic re- sites of the genome (Feng et al., 1999; Schubeler et
combination was used extensively to knock out al., 2000; Baer and Bode, 2001).
homeotic genes (Herault et al., 1998). It can poten- The improvement of the expression system based
tially be used to efficiently target the introduction on the control of transgene expression by tetracy-
of foreign genes in embryos. cline is also quite significant (Forster et al., 1999;
Freundlieb et al., 1999).
Transgenesis is and will remain limited by theo-
4. Conclusions and perspectives retical and technical problems. One limitation cer-
tainly comes from the fact that the gene transfer
Since the first transgenic mice were born in 1980, into an animal is the come back of an isolated gene
L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145 160 155
to the complexity of a whole living organism. It is circular DNA structure (Kelleher et al., 1998)
disappointing that almost 30% of gene knock-out would be of great help for transgenesis. Such
do not result in significant phenotypic modifica- vectors are theoretically capable of giving an ex-
tions. The conditional gene knock-out and the tremely high yield of transgenic animals. On the
possibility to use RNAi to inhibit gene expression other hand, the expression of transgenes is expected
will certainly reduce the proportion of failures in to be high and reliable due to the fact that several
this field of research. copies of the vectors may be present in each cell and
Genes obviously contain multiple signals in their that the foreign DNA is not integrated and thus not
transcription regulation regions but also in their subjected to chromatin environment.
transcribed regions. Only part of these signals are Gene mutation in situ or in test tube may perhaps
known. Gene constructions may result in the asso- be improved by the use of ribo-deoxyribo-oligonu-
ciation of these signals in an inappropriate manner cleotides (Graham et al., 2001) or of the bacterial
leading to alternative splicing, mRNA destabiliza- Rec A system (Pati, 1998).
tion etc. These kind of artefacts cannot be pre- The different tools to inhibit gene expression,
vented as long as all the mechanisms of gene oligonucleotides forming a triple helix with DNA
expression are not better understood. or RNA, antisense RNA and ribozymes, are quite
An increasing number of data indicate that attractive but the optimal conditions of their use
transgenes but also in some cases genes are ex- remain to be found.
pressed in a variegated manner. This mosaicism of The application of animal transgenesis for basic
expression is considered to be due in number of research started in 1980 when the first transgenic
cases to the presence of retrotransposons in the mice obtained by DNA microinjection. Indeed, the
vicinity of the genes or transgenes. The retrotrans- first transgenes were not expressed although it was
posons are very abundant in mammalian genomes perfectly active when transfected in cultured cells.
and most of them are inactivated by DNA methy- About one decade later this initial failure met an
lation. During embryo development, retrotranspo- explanation which led to the concept of LCR
sons as genes are demethylated and thus activated insulator and chromatin opener. This pioneer ex-
and later selectively remethylated. It appears more periment revealed that some essential mechanisms
and more that remethylation of transposons is of gene expression can be studied only in vivo.
partly stockastic leading to an unpredictable activa- More generally, it is clear that the availability of
tion or inactivation of their expression. It is admit- the majority of human and laboratory animal genes
ted that transposons can stimulate or inhibit the identified by systematic genome sequencing or
expression of the neighbor genes. Hence, genes and systematic chemical mutation in mouse (Brown and
transgenes may be subjected to the stock astic Balling, 2001) will require the frequent implemen-
activation of transposons. This phenomenon is tation of transgenesis to study their function. Gene
probably the most prominent epigenetic mecha- addition, gene replacement, gene mutation possibly
nism influencing gene and transgene expression by the use of RDO or of the Rec A bacterial system
(Whitelaw and Martin, 2001; Symer and Bender, and gene trap (Cecconi and Meyer, 2000; Medico
2001; Rakyan et al., 2001). One of the function of et al., 2001) will be more and more used for that
insulators seems to reduce to some extent the purpose.
impact of epigenesis on gene and transgene expres- It is admitted that up to 5000 genes have been
sion. knocked out by homologous recombination in
Improvements of transgenesis techniques are mouse. These experiments include the study of gene
expected. A better understanding of the phenom- function but also the generation of models for the
ena which occur during cloning by nuclear transfer study of human diseases. Mouse is presently the
should progressively contribute to enhance the most utilized species for this purpose. It is now
efficiency of transgenesis by this method. more and more evident that Drosophila and human
Autonomously replicating vectors based on ar- share many of their genes and interestingly more
tificial chromosomes (Voet et al., 2001) or on than 60% of the genes implicated in human diseases
156 L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145 160
have Drosphila orthologues (Bernards and Hari- cally modified plants already available for animal
haran, 2001). Gene manipulation is easier in and human consumption. This is obviously due to
Drosophila than in mouse. It is therefore expected the technical hurdles and the cost of transgenesis
in farm animals. The possibility to use cloning
that Drosphila will be more systematically used
for the study of human diseases. The same rea- technique for gene addition and replacement in
farm animals has opened new avenues for
soning can be applied to another invertebrate,
breeding.
Caenorhabditis elegans, the genome of which has
Among the projects in development, the follow-
also recently been fully sequenced. Other species,
ing are worth being mentioned.
namely rat (Charreau et al., 1996), rabbit (Fan et
Pigs and sheep expressing growth hormone or
al., 1999) and pig will be more frequently used in
IGF1 genes have an amplified muscle develop-
the future to study human diseases. Several argu-
ment with no alteration of their health. Several
ments converge in this direction. These mam-
fish species have significant or considerable
malian species have in some cases metabolic
growth acceleration when they express a growth
properties closer to human than mouse and
hormone transgene. Their use in aquaculture
surgery is also more easily performed on larger
awaits for the design of breeding conditions pre-
animals. The fact that gene replacement by ho-
venting any dissemination of the transgenesis in
mologous recombination will probably become a
wild water. Indeed, a study using medaka as a
reality in the coming years in theses species via the
model revealed that the GH transgene might have
cloning technique is an additional argument.
deleterious and long term effects on wild fish
The vectors to induce gene mutation by ho-
(Muir and Howard, 1999).
mologous recombination are being improved and
Transgenic mice expressing the lysostaphin gene
they tend to mimic human diseases in a more
in their mammary gland have become resistant to
subtle manner (Petters and Sommer, 2000). A case
infection by Staphylococcus aureus (Kerr et al.,
in point is in the field cancer. A recent work
2001). Transgenic farm animals could be pro-
showed that some vectors allow the k-ras gene to
tected from mastitis by the same transgene. Num-
be mutated in a sporadic manner and in single
ber of animal diseases are expected to be
cells in the adult animal. This elegant approach
prevented by the action of various transgenes
reflects more precisely the natural genetic alter-
(M�ller, 2001).
ations which lead to the development of a tumor
Transgenic mice expressing a transdominant
(Berns, 2001; Johnson et al., 2001).
negative myostatin gene show an increased muscle
After the birth of the first transgenic mice ex-
development after birth mimicking the culard
pressing their transgene, the rat human growth
trait. This transgene might enhance meat produc-
hromone gene, it was suggested that transgenic
tion in farm animals (Yang et al., 2001).
animals could become an industrial source of
Transgenic mice and pigs expressing a phytase
recombinant proteins for pharmaceutical use.
gene in the salivary gland (Ward, 2001; Golovan
This hypothesis is close to become a reality
et al., 2001a,b) or intestine have become capable
(Houdebine, 2000).
of digesting phytic acid present in plants. This
The increasing shortage of organs and cells for
leads to a quite significant reduction of phosphate
transplantation to humans inclines to use these
release in environment and to accelerated growth
biological materials from pig. This can become a
of the animals.
reality only when the rejection mechanisms will be
The transfer of genes involved in wool synthesis
understood and controlled and when expression
into sheep has modified fiber composition. The
of endogenous retrovirus genomes will be inhib- aim of the study is to enhance wool growth but
ited. Transgenic animals play a central role in this
mainly to improve wool properties (Bawden et al.,
medical project (Houdebine and Weill, 1999).
1999).
The use of transgenesis to improve animal Gene addition and replacement are potent tools
breeding has received little attention so far. This is to optimize milk composition for animal and hu-
a glaring contrast with what happens with geneti- man consumption (Houdebine, 1998). The secre-
L.-M. Houdebine / Journal of Biotechnology 98 (2002) 145 160 157
Bulfied, G., 2000. Farm animal biotechnology. TIB Tech. 18,
tion of a lactase in the milk of transgenic mice
10 13.
reduced lactose concentration (Jost et al., 1999).
Capecchi, M.R., 1989. Altering the genome by homolgous
The transfer of a gene coding for a mutated
recombination. Science 244, 1288 1292.
human phenylalanine-free -lactalbumin into cow
Cecconi, F., Meyer, B.I., 2000. Gene trap: a way to identify
allows the production in milk of this protein for novel genes and unravel their biological function. FEBS
Lett. 480, 63 71.
patients suffering from phenylketoneurea (PPL
Chan, A.W., Chong, K.Y., Martinovich, C., Simerly, C.,
Therapeutics, unpublished data).
Schatten, G., 2001. Transgenic monkeys produced by
Many other possibilities can be envisaged in
retroviral gene transfer into mature oocytes. Science 291,
this field (Bulfied, 2000; Ward, 2000). Transgene-
309 312.
Chan, A.W., Homan, E.J., Ballou, L.U., Burns, J.C., Bremel,
sis, including the intentional dissemination of the
R.D., 1998. Transgenic cattle produced by reverse-tran-
transgenes in herds, will remain a heavy and
scribed gene transfer in oocytes. Proc. Natl. Acad. Sci.
costly task in farm animals. A success thus implies
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that relevant genes have been identified to expect
Charreau, B., Tesson, L., Soulillou, J.P., Pourcel, C., Anegon,
a beneficial impact for breeders or consumers.
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Chesn�, P., Adenot, P.G., Viglietta, C., Baratte, M., Bou-
which has been undertaken several years ago of-
langer, L., Renard, J.-P., 2002. Cloned rabbits produced by
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