Relations between animal transgenesis and reproduction

363Reprod. Nutr. Dev. 45 (2005) 363–376© INRA, EDP Sciences, 2005DOI: 10.1051/rnd:2005027

Review

Relations between animal transgenesis and reproduction

Louis-Marie HOUDEBINE*

UMR Biologie du Développement et Reproduction, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex, France

Abstract – Transgenesis has become an essential tool for the study of gene expression mechanismsand functions. Transgenesis is also more and more used for biotechnological applications such asthe study of human diseases, the adaptation of pig organs to humans, the production of pharmaceuticalproteins in milk and likely in the future for the improvement of animal production. The use oftransgenesis relies on the efficiency of gene transfer. New tools have been recently designed toimprove gene transfer. The methods of gene transfer are highly dependent on the techniques of animalreproduction. Conversely, the need to improve transgenesis urges researchers to study some of thekey steps in reproduction and to find new techniques for gene transfer. This paper summarises therecent data and the perspectives offered by animal transgenesis.

transgenesis / animal production / biotechnological applications

1. INTRODUCTION

The first demonstration about 30 yearsago that it was possible to isolate genes, torecombine DNA fragments in vitro and tointroduce native or recombinant genes intobacteria was rapidly followed by theexpression of foreign genes after transfec-tion into animal cells. The idea of reintro-ducing a gene in the genome of animalsappeared logical, quite attractive but noteasily feasible in a short term.

The demonstration in 1980 that foreigngenes could be integrated into the mousegenome and transmitted into progeny was

a great technical and symbolic event for thescientific community. This initial successwas repeated and followed two years laterby the generation of transgenic mice har-bouring foreign growth hormone genes andshowing a greatly enhanced growth. Theproof was given for the first time that for-eign genetic information could be expressedand induce specific phenotypic effects.Broad perspectives for basic research andbiotechnological applications in the medi-cal and agronomical fields appeared open.The fact was perceived not only by the sci-entific community but also by citizens. Yet,it took years before a number of biologists

* Corresponding author: [email protected]

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364 L.-M. Houdebine

not familiar with molecular approachesrealised that they were living a technical andmethodological revolution. Until the end ofthe 1980’s, some biologists remained con-vinced that transgenic mice became giantbecause they received growth hormonegenes from species (rats and humans) largerthan themselves. Similarly, it took timebefore some biologists admitted that a moremolecular approach including transgenesiswas highly beneficial to the study of bio-logical functions.

In 1985, transgenic rabbits, pigs andsheep were obtained indicating that thetechniques originally defined for mice couldbe extended to other mammals. In 1986, thefirst transgenic fish were obtained usingDNA microinjection into the cytoplasm ofembryos rather than the pronuclei which arenot visible in these species.

The first success of transgenesis appearedsooner than anticipated. It was the result ofa multidisciplinary approach including embry-ology, biology of reproduction, molecularbiology and genetic engineering. Transgen-esis is now achieved in mammals includingfarm animals and in a near future in pets, aswell as in vertebrates and invertebrates.About 25 animal species are presently usedto develop transgenic lines for basicresearch or applied purposes (Fig. 1).

Since the invention of agriculture andbreeding, genetic selection relies on theobservation of living organisms followedby the preferential reproduction of some ofthem. The same became true more recently

for laboratory animals used to study biolog-ical and gene functions as well as humandiseases.

Modern biology relies more and more onthe systematic generation of lines of livingorganisms having phenotypical character-istics followed by the identification and thestudy of the genes responsible for theobserved biological properties of these indi-viduals. A broader biodiversity is currentlybeing created by inducing random muta-tions using chemical mutagens or irradia-tion. This procedure is now being extendedto a mammal, the mouse, giving rise to rel-evant models but remaining highly impre-cise. In plants, interspecies crossing iscurrently leading to the generation of newvarieties and even to new species.

Transgenesis offers unprecedented pos-sibilities. It is more precise, since essentiallyonly one or a few known genes are beingmodified in a genome and foreign genes canbe introduced in a given species (Fig. 2).

Transgenesis is a more and more usedtechnique. It offers the possibility to studyand use the newly discovered genes whichare becoming more and more numerouswith the systematic sequencing of genomes(Fig. 3).

From the beginning, the success of trans-genesis is highly dependent on the controlof reproduction. Each group of species raisesdifferent problems, specially for gene transfer.It is noteworthy that gene transfer implies,according to species, superovulation, invitro maturation and fertilisation including

Figure 1. The different key steps in animal transgenesis.

Transgenesis and reproduction 365

ICSI, cloning by nuclear transfer, genera-tion of chimerae by blastomere transfer,gene transfer into seminal tubules, collect

and reimplantation of spermatocytes aftergene transfer, embryo culture and embryotransfer.

Figure 3. Consensus structure and the different utilisations of eucaryotic genes.

Figure 2. Comparison of classical selection based on sexual reproduction with transgenesis. Inclassical selection, the experimenters ignore the nature of the selected gene of interest and of thecoselected genes which may have deleterious effects. In transgenesis, the foreign gene is knownand brings a single genome modification which may be studied in detail.

366 L.-M. Houdebine

Clearly, a progress in the techniques ofreproduction may facilitate transgenesis.Conversely, transgenesis is quite difficultto achieve by DNA microinjection into pro-nuclei in some species such as ruminants.This urged researchers to develop alterna-tive techniques. The birth of Dolly thesheep was achieved with the aim of facili-tating gene transfer. Similarly, sperm manip-ulation is being improved not only to describespermatogenesis and fertilisation but alsoto tentatively offer new ways of gene trans-fer. Transgenesis thus accelerates somespecific studies in biology of reproduction.More marginally, reproduction may indi-rectly take advantage of transgenesis. Oneexample is the preparation of large amountsof bovine FSH and possibly of human FSHand other hormones in the milk of trans-genic animals.

Animal transgenesis is presently facingthree major problems (i) gene transfer togenerate transgenic lines, (ii) reliableexpression of transgenes, (iii) interpreta-tion of the data obtained with transgenicanimals. The first two problems are essen-tially technical whereas the third is inherentto the approach which consists of returninga simple gene back to its natural complexity.

This paper is a brief survey of animaltransgenesis. A more detailed descriptionof the techniques and applications of trans-genesis has been reported in a book [1].

2. TECHNIQUES OF GENE TRANSFER

Unless genes are included in viral parti-cles, they cannot penetrate spontaneouslyinto cells. Their large size, their ionic chargeand their sensitivity to surrounding DNAsepreclude spontaneous DNA transfer intocells and embryos. Various methods havebeen designed to transfer foreign DNA. Beforedescribing these methods, it is important toconsider the mechanisms involved in for-eign DNA integration into genomes.

2.1. The fate of foreign DNA

When added into a nucleus, a linear for-eign DNA is circularised, randomly cleavedand associated according to a homologousrecombination process, generating per-fectly matched polymers in which genes arein tandem arrays.

When added into cytoplasm (after trans-fection, electroporation or microinjection),DNA fragments are randomly associatedforming tandem or head to tail polymerswith some rearrangements.

Part of the DNA migrates to the nucleus.Foreign genes in the nucleus can be tran-

siently expressed and are destroyed duringthe next cell replication unless they are inte-grated.

Foreign DNA are randomly digested byexonucleases which generate single strandends capable of recognising similar but usu-ally not identical sequences in the genome.This leads to random integration of the for-eign DNA. Alternatively, foreign DNA canreplace an identical host DNA region accord-ing to a homologous recombination process.This leads to host gene inactivation (knockout) if the foreign gene construct is an inac-tive gene. This leads to the expression of amutant or a quite different gene (knock in)if the foreign gene construct is a functionalgene.

Homologous recombination is a rareevent (0.1–1%) of heterogeneous recombi-nation. Gene replacement must therefore beachieved in cells which are selected and fur-ther used to generate an embryo.

2.2. DNA microinjection

DNA can be microinjected into the pro-nuclei of mammals only. In other species,the vitellus and the shell do not allow a vis-ualisation of the pronuclei. Microinjectionmust then be achieved into the cytoplasm.About 1 000–5 000 and 1–20 million copiesare injected into the pronuclei and the cyto-plasm respectively.


For unknown reasons, the integrationrate is highly variable according to species.Up to 1–3% of microinjected embryos maybecome transgenic mice whereas the rate islower for rabbits, rats, pigs and extremelylow in ruminants. Integration essentially doesnot occur in chicken, xenopus and medakaembryos (although it is relatively high inother fish species such as salmonids).

In order to improve integration, the for-eign genes may be inserted into trans-posons. For this purpose, the integrase geneof the transposon is deleted to make spacefor the foreign genes and to prevent therecombinant transposon to disseminate inthe genome autonomously. To become inte-grated, the recombinant and defective trans-poson must be complemented by exogenousintegrase either comicroinjected with thetransposon or synthesised from a plasmidharbouring the integrase gene.

A variety of transposons are being imple-mented according to species. Transposon Pis extensively used to generate transgenicDrosophilae. The transposons SleepingBeauty and piggy Bac are used for a numberof species [2] and for the silk worm [3]respectively. Other transposons are used forvarious species of insects.

Transposons are efficient and safe butthey can harbour no more than 3–4 kb offoreign DNA.

2.3. Use of retroviral vectors

Retroviral vectors are extensively stud-ied to transfer genes to somatic cells ofpatients. These vectors have met some suc-cess particularly to allow immunodeficientchildren to leave their protective bubbles.These vectors have been recently improvedusing lentiviral genomes and an envelopefrom the vesicular somatitis virus. Highconcentrations of particles can be obtained.These vectors can infect all cell types andthey transfer their genetic material to thehost genome in quiescent as well as in rep-licating cells [4].

Foreign genes inserted into lentiviralvectors are also generally not silenced asopposed to those transferred by conven-tional retroviral vectors.

These vectors have proven highly effi-cient in generating transgenic mice [4], pigs[5], cows [6], chickens [7, 8] and sheep [9].

For unknown reasons, lentiviral vectorshave to be injected into the oocyte in orderto generate transgenic cows whereas injec-tion into a one cell embryo is preferable inmost other species (Fig. 4).

Lentiviral vectors are much more effi-cient than classical microinjection in somespecies such as ruminants or chickens. Onelimitation is that lentiviral vectors can har-bour at most 8.5 kb of foreign DNA. Thismay be hardly enough in some cases.

2.4. Use of sperm cells to transfer genes

Experiments carried out more than onedecade ago showed that sperm incubated ina DNA solution can transfer the foreigngene into the oocyte during fertilisation.This extremely simple technique gave birthto transgenic animals of different species.The method which originally appeared poorlyreproducible has been greatly improved byeliminating the DNAse which is abundantin seminal plasma and on the sperm surface[10].

This approach has been improved by ini-tially degrading the sperm membrane. Thisallows DNA to penetrate abundantly intothe sperm but precludes spontaneous fertili-sation. ICSI (intracytoplasmic sperm injec-tion) is then required to fertilise oocytes withthe damaged sperms. This technique wasinitially defined to generate transgenicxenopus. It has been successfully extendedto other species. Interestingly, the mostrecent publications indicate that the effi-ciency can be greatly improved by modify-ing the protocol and also that DNAfragments as long as 200 kb can be trans-ferred into embryos with a good yield andwithout any degradation of the DNA. Thissuggests that ICSI could be used more

368L

.-M. H

oudebine

Figure 4. Gene transfer into oocyte, embryo or primordial germ cells using lentiviral vectors. The viral proteins are provided to the defective viralgenome harbouring the foreign gene by a transcomplementing cell.


broadly to generate transgenic animals in avariety of species [11–13].

An elegant method which is being usedby one group consists of incubating spermwith an antibody which specifically recog-nises a surface antigen. This antibody alsohas in its C terminal part a stretch of basicaminoacids that spontaneously binds DNA.The sperm-antibody DNA complex wasused to generate transgenic mice, pigs andcows with good efficiency [14, 15].

Foreign DNA can be transferred directlyinto sperm precursors by injecting DNA-transfectant complexes into seminal tubules[16]. Alternatively, sperm cell precursorsmay be collected, transfected in vitro andreimplanted into recipient testis [17, 18].This allows gene addition and potentiallygene replacement using homologous recom-bination.

2.5. Use of chimaeric animals

One way to generate living geneticallymodified animals with cells in which geneaddition or gene replacement has beenachieved consists of using the capacity ofpluripotent cells to participate to develop-ment after having been injected into a blas-tocyst. In the best cases, the resultinganimals are mosaic for the transgene whichis present in germinal as in somatic cells.This allows the further generation of linesof animals homozygous for the geneticmodification [19].

Although relatively laborious, thisapproach allowed to knock out more than5 000 genes in mice. This technique pro-vides researchers with a wealth of informa-tion. In a number of cases, the data obtainedwith these knock out animals cannot be eas-ily exploited since the homozygous animalsare not viable or show no phenotypic mod-ification.

This elegant method is still restricted totwo lines of mice. In other lines as in otherspecies, despite an intense effort, pluripo-tent cells capable of transmitting theirgenome to progeny have not been estab-

lished. The reason why the two mouse linesallow the use of ES (embryonic stem cells)to generate chimaeric animals with germi-nal transmission is not known. This raisesthe question of knowing what is a pluripo-tent ES cell. Several genes involved in themaintenance of the pluripotent state of cellshave been identified and others are understudy. These genes are transfected into cellsof early embryos from mice and other spe-cies with the hope that this will allow theestablishment of ES cell lines and their useto knock out genes.

2.6. Use of cloned animals

To circumvent the lack of utilisable EScells, the cloning technique has been imple-mented. In practice, it consists of adding aforeign gene or replacing an endogenousgene by homologous recombination in fetalsomatic cells and using their nuclei to gen-erate cloned transgenic animals.

Gene addition was achieved soon afterthe birth of Dolly. This approach has beenadopted by all the groups working withruminants. Indeed, although laborious, genetransfer is easier and quite significantlymore efficient by cloning than by classicalmicroinjection in these species.

Gene replacement by homologous recom-bination can be presently achieved in spe-cies other than mice only by implementingthe cloning technique. This remains a diffi-cult task for several reasons. Homologousrecombination is less frequent in somatic thanin pluripotent cells. On the contrary, theselection of the cells in which gene replace-ment has occurred is a relatively long proc-ess implying the use of antibiotics. Theseconditions of culture, alter cells in anunknown manner rendering a successfulcloning less likely.

Gene replacement by cloning was ini-tially achieved in sheep [20] and soon afterin mice. Interestingly, the galactosyl trans-ferase gene in the pig has been knocked outin this manner with no major difficulty [21,22]. For an unknown reason, pig cells are

370 L.-M. Houdebine

less sensitive than ruminant cells to factorswhich reduce cloning efficiency.

A recent study has shown that it is pos-sible to knock out both alleles of two genesin the same cow. To reduce the artefacts ofcloning, the authors of this work knockedout a first allele and used cells of the result-ing foetus to knock out the second allele.This protocol was followed to inactivateboth alleles of a second gene [23]. The pas-sage to the foetal state gives the best chanceto eliminate the clones that will have theless chance to survive.

Interestingly, one of the knock out geneshere codes for PrP, the protein which playsa major role in bovine prion disease.

3. VECTOR DESIGN FOR TRANSGENE EXPRESSION

Genes contain multiple signals in theirtranscribed as in their promoter regions.These signals are not all known and theirassociation in gene constructs leads fre-quently to poorly active transgenes.

After more than two decades, empiricalrules to design vectors which express trans-genes efficiently have emerged. Transgenes

are poorly expressed when they contain nointrons, when they are rich in CpG regionsand when they are integrated in tandemarrays [24]. Strategies of gene constructionmust therefore take these observations intoaccount to augment the chance of transgeneexpression.

It is now clear that in eucaryoticgenomes, genes are generally clusteredforming loci which are bordered by insula-tors. Insulators which may be located 10–50kb or more from the genes have severalknown functions and likely others whichremain to be discovered.

They contain enhancer blockers whichprevent the gene regulators of a locus tointeract with the genes of the neighbouringloci. Insulators also contain elements capa-ble of locally maintaining chromatin in anopen configuration (euchromatin). This isachieved with chromatin factors whichfavour histone hyperacetylation and pre-vent DNA methylation (Fig. 5).

The existence of insulators was revealedby the fact that some patients suffering fromthalassemia had a non-mutated β-globingene and promoter but a deletion of a farupstream region. Moreover, this region

Figure 5. The mechanism of action of insulators. Remote regulatory elements are concentrated ina hub thanks to the formation of loops. The factors concentrated in the hub maintain an openchromatin conformation by hyperacetylating histones. This allows an efficient gene expression.


known as LCR (locus control region) orinsulator allows a reliable expression of theβ-globin gene in transgenic mice. The samegene remains silent when it is not associatedwith the LCR.

In about 20 cases, long DNA genomicfragments allow a reliable expression of thetransgene they contain. These long DNAfragments may be used as vectors to expressassociated foreign genes. Likely, in a fewyears, BAC vectors containing long DNAfragments allowing efficient transgeneexpression in a variety of cell types will beavailable. It is also conceivable that the ele-ments forming insulators will be identifiedand concentrated into compact structurescapable of preventing interactions of chro-matin and transgenes. This would allowreliable transgene expression but also nodeleterious activation of host genes (such asoncogenes) by the enhancers added in thetransgene constructs. In the mean time,fragments of insulators may improve trans-gene expression [25].

3.1. Use of RNAi to knock down genes

Fortuitous observations have shownthat, unexpectedly, double strand RNAinhibit much more potently mRNA sharingthe same sequence than single strand RNA.The essential of the involved mechanismshas been deciphered. Long double strandRNA are randomly cleaved into 21–23 bpfragments which are associated with a pro-tein complex which allows specific recog-nition and degradation of the mRNA havingsimilar sequences (Fig. 6) [26, 27].

This small RNA known as interferingRNA (siRNA or RNAi) may be transfectedto cells or synthesised by appropriate vectors.

Transgenic mice in which the ski genehas been knocked down by a RNAi showsimilar biological characteristics to thoseobserved in mice in which the ski gene hasbeen knocked out by homologous recombi-nation [28].

Double strand RNA show a more or lesspotent interfering effect according to thetargeted sequence consensus sequences

recently discovered [26]. The extensive useof RNAi in cells, tissues and transgenic ani-mals to inhibit endogenous or viral genesappears more and more attractive.

The advantage of gene knock down overknock out is its relative simplicity, its flex-ibility and its reversibility.

Vectors allowing an inducible and revers-ible induction of RNAi synthesis in trans-genic animals are under study and should beavailable in the coming years.

Short double strand RNA can also specif-ically inhibit gene expression by inducing aDNA methylation in the promoter region.This phenomenon known as TGS (tran-scriptional gene silencing) which is differentfrom mRNA degradation (PTGS: posttran-scriptional gene silencing) is irreversibleand even transmitted to progeny [29, 30].

The expression of a gene may be blockednot only at the DNA or mRNA levels butalso at the protein level. Overexpression ofa non-secreted antibody in transgenic ani-mals may inhibit the cellular protein recog-nised by the antibody [31].

Another possibility consists of overex-pressing a transdominant protein. A recentstudy may exemplify this approach. Asecretion of the soluble region of pseudor-abbies virus responsible for Aujeszky dis-ease in transgenic mice used as modelsprevents infection by the virus. The virus isunable to make a distinction between thenormal receptor on cells and the solublebinding site. The virus is thus trapped by theoverexpressed soluble receptor and becomesunable to infect cells and animals. Genera-tion of transgenic pigs resistant to Aujeszkydisease is now conceivable [32].

This approach is theoretically possibleeach time a transdominant negative proteinis available.

3.2. Special vectors for transgene expression

Transgenes containing regulatory ele-ments of animal genes may be regulated in

372 L.-M. Houdebine

a specific way in vivo. Yet, in some circum-stances it may be important to induce ordeinduce a transgene using unducers notacting on host genes.

Several systems are available to reachthis goal. The most popular is based on theuse of tetracyclin or analogues to induceexpression of transgenes to which a controlelement sensitive to a tetracyclin repressor

has been added. In practice, one transgenicline harbours the gene coding for a fusionprotein containing the tetracyclin repressorand an animal gene activator. A secondtransgenic line harbours the gene of interestunder the control of a tetracyclin repressorregulatory element. Tetracyclin added tothe water of hybrid animals harbouring bothtransgenes induces the gene of interest in a

Figure 6. The mechanism of the inhibition of gene expression using interfering RNA (RNAi).Chemically synthesised small RNAi targeting a given cellular mRNA may be added to cells orgenerated in cells or animals by transgenes coding for these RNAi. The targeted RNA arespecifically knock down.


reversible manner. In some circumstances,this method greatly improves the relevanceof the transgenic models. It may render pos-sible the study of a transgene which codesfor a protein too deleterious for cells to beexpressed permanently.

A family of vectors has been designed totrap unknown genes or promoters having animpact on a biological function. These vec-tors may contain a reporter gene devoid ofan intron. The random integration of sucha vector is performed in ES cells furtherused to generate transgenic mice. In somecases, the integration of the vector is fol-lowed by expression of its reporter gene andthe alteration of a biological function in ani-mals. This may mean that the integration ofthe vector occurred in a host gene whichplays an important role in the altered func-tion. The targeted gene may be identifiedusing the reporter gene as a marker.

Gene trapping is a somewhat laboriousmethod which may lead to the establish-ment of unpredictable correlations betweenthe action of a gene and a biologicalfunction.

4. THE APPLICATIONS AND THE FUTURE OF ANIMAL TRANSGENESIS

A vast majority of transgenic animals areused to study gene action and function. Thesystematic sequencing of an increasingnumber of genomes renders the use of trans-genesis still more necessary.

DNA integrated into an animal genomeis quite stable and transmitted to progenyessentially without any modification. Trans-gene expression is also relatively reproduc-ible in the different animals of a given line.It remains that up to 7–20% of host genesare interrupted and inactivated by the inte-gration of a foreign gene [33]. Gene target-ing and more generally reliable expressionvectors would greatly reduce most of the

artefacts due to the random integration offoreign genes.

Transgenic animals are a potent tool togenerate models for the study of human dis-eases [34]. Mice which do not live morethan two years have a negligible chance tospontaneously develop Alzheimer disease.The transfer of three human genes into miceled to the creation of a relevant model forthe study of the disease and the evaluationof new pharmaceuticals.

The relevance of the models is highlydependent on an appropriate transgene expres-sion as well as on gene replacement byhomologous recombination. The mouse isthe most frequently used species for thispurpose but in some cases other species arerequired. This is the case when surgicaloperations such as organ grafting is beingachieved. Rats or rabbits are then moreappropriate. In some cases, animals likerabbits are preferred to mice for biologicalreasons. Indeed, lipid metabolism is moreeasily studied in rabbits than in mice sincethe former are closer to humans than the latter.

More than 50 lines of transgenic mice areavailable and can be purchased as other lines.

Experimenters wish to use more andmore precise models. This implies thattransgene expression be well-controlledand that gene replacement can be performedin several species. Technical progress in genetransfer and transgene expression is stillneeded to reach this goal.

It becomes more and more likely that pigorgans and cells will be some day used fortransplantation to patients. Quite encourag-ing results have been recently obtained. Theknock out of the galactosyl transferase genein the pig resulted in the absence of the mostpotent antigen at the surface of pig cells.This allowed kidneys from the transgenicpigs to be maintained healthy in experimen-tal primates for at least two months.

Production of pharmaceutical proteins inthe milk of transgenic animals is progres-sively becoming a reality. One protein,human antithrombin III, is currently underevaluation by the European agency EMMA.

374 L.-M. Houdebine

Many other proteins and mainly chimaeric;humanised or human monoclonal antibodiesare expected to be produced at a moderatecost in the coming decade and later [35].

Applications of transgenes to improveanimal production are still rare and none ofthese new products are on the market. Thisis clearly due to the difficulty and the costof generating transgenic farm animals. Therecent technical progress and particularlythe implementation of cloning to add orreplace genes offers quite attractive possi-bilities.

Among the project in course, a few ofthem can be mentioned. Transgenic fish,and mainly salmons, having an acceleratedgrowth are on the way for authorisation tobe used for human consumption. The prob-lem of the possible dissemination intooceans has not been solved yet [36, 37].

Pigs expressing bacteria phytase in theirsaliva reject 75% less phosphate into theenvironment leading potentially to a signif-icant reduction of pollution [38].

Goats or cows expressing an antibacteriaprotein such as human lysozyme, humanlactoferrin or lysostaphin in their milk areexpected to be less sensitive to mastitis.Their milk resists to corruption by bacteriainfection and it might be used by personssuffering from bacteria infection [39, 40].

The struggle against diseases appearsparticularly interesting and technically fea-sible. Animals genetically resistant to dis-eases may need a lower use of pharmaceuticalcompounds including antibiotics. Theirbreeding may be facilitated and less costly.They may also reduce the possible transferof animal diseases to humans. The resist-ance to Aujeszky disease appears as aninteresting example.

Attempts to improve the quality of ani-mal products appear attractive. This is thecase for pigs expressing a desaturate genefrom spinach which enhances the propor-tion of now saturated lipids [41].

A long study is still required to validatethese lines of animals.

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Relations between animal transgenesis and reproduction