genomics and ethics: the case of cloned and/or transgenic animals · genomics and ethics:...

Comparative and Functional GenomicsComp Funct Genom 2003; 4: 26–30.Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/cfg.243

Conference Review

Genomics and ethics: the case of cloned and/ortransgenic animals

Beatrice de Montera*Laboratoire de Biologie du Developpement et Biotechnologies, INRA, Domaine de Vilvert, 78 352 Jouy-en-Josas cedex, France

*Correspondence to:Beatrice de Montera, Laboratoirede Biologie du Developpementet Biotechnologies, INRA,Domaine de Vilvert, 78 352Jouy-en-Josas cedex, France.E-mail: [email protected]

Received: 7 September 2002Accepted: 29 November 2002

AbstractThe point of the present study is to illustrate and, if possible, promote the existing linkbetween genomics and ethics, taking the example of cloned and transgenic animals.These ‘new animals’ raise theoretical and practical problems that concern appliedethics. We will explore more particularly an original strategy showing that it ispossible, starting from philosophical questioning about the nature of identity, to usea genomic approach, based on amplification fragment length polymorphism (AFLP)and methylation-sensitive amplification polymorphism (MSAP) detection, to provideuseful tools to define more rigorously what cloned animals are, by testing their geneticand epigenetic identity. We expect from the future results of this combined approachto stimulate the creativity of the philosophical and ethical reflection about the impactof biotechnology on animals, and to increase scientific involvement in such issues.Copyright 2003 John Wiley & Sons, Ltd.

Introduction

We aim to demonstrate the existing link betweengenomics and bioethics by a reflection of appliedethics that relies on the use of genomic tools.

Taking the case of cloned and/or transgenic ani-mals, one can pose a philosophical question aboutthe identity of such animals, identity being definedphilosophically as a permanence or a resistance toexternal change. However, this definition is not suf-ficient to explain the particularity of these ‘newbeings’ [9]. A genomic approach that would anal-yse the link between DNA structure and geneexpression, by comparing genomes, could supplyuseful tools to clarify our understanding aboutcloned animals. It should be very interesting tobuild, on a scientific basis, some new philosoph-ical questioning that could lead to the explorationof new pathways in applied ethics.

The cloning technique is difficult to classifybecause cloned animals are generally not consid-ered as genetically modified. We can only notethat cloning is a form of asexual reproduction thatis unnatural in mammals. Transgenesis, by con-trast, consists of a manipulation of the genome.

Therefore, theoretical questions about the identityof cloned animals and transgenic ones are quite dis-tinct, even if these techniques are not necessarilyseparate from a pragmatic point of view becauseusing cloning via cell transformation remains themost profitable way of producing transgenic ani-mals in cow and goat. The existence of clonedmammals obtained by nuclear transfer (which didnot exist 7 years ago), in our case bovine clones,poses the problem of genetic identity in a differentway to that of inbred animals. The initial purposeof cloning was to create genetically identical ani-mals as so-called ‘good experimental models’, butthe question is, are they truly genetically identical?

In addition, given that cloned animals have a dif-ferent methylation pattern, one can suggest that itmight be simplistic to assume that genetic identityis the only form of identity to examine. To appre-hend the issues around identity in an original butscientific way, it is possible to use genomic toolssuch as AFLP [17] to test both genetic identity andmethylation differences. Having scientific tools tostudy the identity of cloned animals should allowfor improvement in the definition of these ‘newanimals’, and therefore stimulate a more concrete

Copyright 2003 John Wiley & Sons, Ltd.

Genomics and ethics: cloned/transgenic animals 27

reflection integrating their scientific, social and eth-ical status.

On the other hand, transgenesis poses the ques-tion of the physical resistance between species.This limit to manipulation could point to the exis-tence of a species-specific identity.

These reflections lead us to two pragmatic ques-tions: what is the utility of creating such new indi-vidualities, and what is the responsibility of thescientific community in such matters?

Materials and methods

How can we go about answering the question of theidentity of clones in a scientific way? If we considerthat clones have no pathology, can we assert thatthey are normal and genetically identical, as theyare expected to be? If we assume that sequencingthe entire genome of each bovine clone is not fea-sible, we can handle the question of identity byusing the AFLP technique [1] to study the geneticvariability of normal clones. In order to find someanswers to the question of the identity of these‘new animals’, we are comparing the genomes ofbovine clones derived from the same batch of cellcultures at the French National Institute of Agro-nomic Research (INRA). AFLP has not been triedon twinned animals, therefore we do not yet havean estimate of their degree of identity. Twins appearnaturally (post-fertilization) by a process of sponta-neous embryonic scission; clones, by contrast, arecreated by an artificial process before development,which avoids fertilization [13]. Would genomesoriginating from the same somatic genotype beidentical in terms of their linear DNA structure,as are twins? The answer is normally ‘yes’ fortwins (except in cases where spontaneous muta-tions occur), but needs to be tested particularlyregarding clones, since clones derive from differentnuclei that are artificially transferred into differentcytoplasms. Would clones and twins be identicalin terms of the function of their genome? Proba-bly not, because of the differential methylation ofgenes in a given local environment. This questionis particularly relevant regarding cloned animalsbecause of the existence of variations in DNA (loci)methylation [11]. The questions of structural andfunctional identity of clones have therefore to beaddressed in order to provide an estimate of thedegree of difference.

DNA from cells obtained from ear skin and cul-tivated during one passage is purified with a DNAextraction kit (Qiagen, San Diego, USA). DNA isthen digested with a rare site enzyme (EcoRI) anda frequent one (TaqI) to generate thousands of frag-ments of an average size of 256 bp. Each end of theamplified fragments is ligated with double-strandlinkers to allow the amplification of all fragmentswith the same pair of primers. Two rounds of PCRare performed with primers containing one to threeadditional bases at each end, in order to reducethe number of possible amplifiable fragments. Thisallows the selective amplification of only a partof the fragment set corresponding to the specificsequence of each combination of primers. To per-mit the detection of amplified fragments on anautomatic sequencer, the rare cutter primer sideis fluorescently labelled. Of the potential 15 × 109

fragments generated by digestion of the bovinegenome, only 50–150 will be detectable using eachprimer combination. The 83 possible combinationscreated by addition of 1–3 base pairs at the endof the amplification primers will provide access to1%, at best, of the sequence information of thegenome in order to check for the presence of pointmutations or rearrangements. The AFLP techniqueis particularly well adapted to the detection of vari-ations within a species. One usually uses the pairof primers that gives the highest number of differ-ences, but our study requires the use of all possibleprimer combinations to obtain the best informationabout clone identity, even if it concerns only 1%of the genome.

If some polymorphisms are detectable, this studyof the frequency of polymorphism in bovineclones will lead to the following question: whatwould the presence of a mutation between twoclones of the same genetic background — samedonor cell — signify? The possible presence ofdetectable mutations [10] in cloned animals couldsignify that these animals are not identical, asthey were supposed to be, and this, in turn, couldchange our way of viewing the cloning technique.The response to the question of knowing whethercloning induces mutations depends on the resultswe will obtain.

The use of a variant of the AFLP technique,using a restriction enzyme sensitive to methylationcalled MSAP [18,19], would be interesting to testthe epigenetic identity of clones by searching foraberrant methylation patterns that, in abnormal

Copyright 2003 John Wiley & Sons, Ltd. Comp Funct Genom 2003; 4: 26–30.

28 B. de Montera

animals, could be linked to problematic cloning andassociated pathologies [7].

Discussion

What are the existing ethical problems andquestioning about cloned and/or transgenicanimals, and what can we expect to learnfrom this scientific approach?

We must realize that clones and transgenic animalsare not technological hypotheses, but concrete real-ities that pose very peculiar problems because theydo not benefit from the protection of any properlegislation or ethics [2]. Until now, they have notbeen taken into consideration by a research ethicsthat has preferred action, instead of control, ofthe biotechnology used. This proposed scientificapproach could help us to more precisely defineand build an ethical status for these ‘existing ani-mals’ which should never be considered only as‘hypothetical’ ones [15]. The pragmatic questionof what to do with cloned or transgenic animalsshould be linked to the question of the legitimacyof their conception and the issues around their liv-ing conditions.

What are the concrete problems that havearisen with existing cloned animals?

First, we must deal with a technical weakness.Until now, bovine cloning, consisting of the fusionbetween a somatic cell and an enucleated oocyte,has had a very low success ratio of 1.5% (numberof calves born : reconstituted embryos [5]). Theproblem is that it is difficult to find an explanationfor such a bad ratio. Hence, there remains a‘grey area’ in the cloning technique that shouldbe clarified, perhaps via scientific studies like ours,which could serve as feedback on the technique.

This fuzzy technical context has heavy conse-quences during the gestation period. As we donot know how the nucleus is re-programmed,we cannot foresee the physiological consequencesduring embryo development [6]. Large offspringsyndrome (LOS) in calves is the main prag-matic issue that scientists have to deal withand that poses ethical problems [20]. The foe-tus is indeed bigger than it should be and isoften characterized by an asynchronous develop-ment of organs, e.g. enormous heart and kidneys,

and small lungs [4]. This syndrome, for whichscientists were not prepared because they havenot solved all the theoretical problems aroundcloning, has resulted sometimes in catastrophessuch as the breakdown of the uterus of some sur-rogate mothers. In this difficult context, the reac-tion of veterinarians and of animals caretakersis empirical.

Until now, decisions have been made using acost vs. scientific interest balance. Creating clonedcalves is expensive — 1.8 times more than fora calf produced using IVF [5] — and of greatscientific interest, so the main purpose is to keepthe foetus alive. This criterion is explicit, but thereis another criterion, which is only implicit, which isthe cost vs. animal suffering balance. In this case,cost symbolizes the global financial and scientificvalue of the calf, and it is compared to the cost ofthe surrogate mother’s suffering. The descriptionof what is usually done underlines the necessity ofmaking decisions within an ethical framework. Inthis case, a conflict must be solved between twoaspects of animal ethics, which are pathocentrism(where the value is the pain of the surrogatemother) and biocentrism (where the value is thelife of the calf). On the other hand, we have tothink about the possibility of creating a new statusfor bovine clones, because of their very particularcare and sanitary conditions, which are much betterthan for other breeding animals.

Using genomic tools can perhaps lead, by clarify-ing the identity of clones, to an explanation for theweakness of the cloning technique and to an avoid-ance of the appearance of abnormalities during thedevelopment. Most importantly, it could allow arigorous reflection about the theoretical and, morespecifically, philosophical issues about the natureof animals created through cloning.

What about the concrete problems that havearisen due to the existence of transgenicanimals?

As we have already said, transgenesis poses a dif-ferent theoretical problem from cloning itself. Oneof the ideas would be to create producers of pro-teins of therapeutic interest, e.g. in milk regardingbovine species. However, it seems largely to bepremature to talk about therapeutic applications,considering the technical problems that currentlyoccur (with the output ratio being extremely low).


Genomics and ethics: cloned/transgenic animals 29

The case of transgenic animals reveals the diffi-culty for a genome to tolerate the transfer of aforeign gene, and we know there can sometimesbe severe consequences of the random insertionof a transgene [16]. Transgenic animals cannot beconsidered as mosaic organisms because they arenot normally made up of cells with different geno-types derived from the same zygote [14], since theyare considered as transgenic only if their cells aretransgenic. A chimera is an organism that con-sists of cells derived from more than one indi-vidual; under this broad definition, transgenic ani-mals could in some ways be considered as geneticchimeras, because their genome is formed frommore than one genome. However, they are not cel-lular chimeras, because chimera cells are usuallyconsidered as being of different genotype and there-fore chimeras are rather a subset of mosaics [14].What could the difference be, then, between trans-genic animals and animals that have undergone hor-izontal gene transfers during evolution, and couldthey be assimilated to chimeras? One answer couldbe that only selected genes, which are useful anddo not cause a disturbance, have not been deletedfrom organisms through evolution after horizon-tal transfer. Through evolution, organisms havetime to react to, and to adapt to, inherited internalchanges, whereas the impact of a transgene, whichis global for the organism, and brutal, does notallow for adaptation or further deletion. We couldsay, therefore, that transgenic animals are not pro-tected by the evolutionary processes of gene lossand gain. The consequences of transgenesis on ani-mals raise the question of specific identity, whilecloning consequences raise the question of individ-ual and genetic identity. However, the existence oftransgenic animals is not only a theoretical subject,and we can consequently ask the practical question:what should be done with transgenic animals oncethey have been created? One can wonder if sci-entists always foresee the pragmatic consequencesof their scientific projects when dealing with engi-neered animals, especially concerning mammals,and a species loved by the public, such as thebovine species.

In order to think about the conceptual issues oftransgenesis and cloning, we can rely on philo-sophical concepts coming from Aristotle, such asform — or essence — and matter; concepts that,once reshaped, can help us to propose an explana-tion of the importance of the principles of identity

and difference for an animal life. We could makethe hypothesis of the transmission through gener-ations of a form that transports a certain degreeof identity and a certain degree of variation [12],corresponding in some way to the transmission ofDNA and to the inheritance of epigenesis. Then thephilosophical question would be: do genetic manip-ulation or cloning affect the essence — or iden-tity — of an animal by introducing an abnormaldegree of identity or difference? In what concernsus, we expect from our scientific study some newingredients to sketch an answer to the question ofidentity concerning cloning.

We have the possibility now of summing up theethical issues concerning this study. We find threequestions with different natures:

1. The first question is an epistemological matter.Six years after Dolly’s birth, there is a realnecessity, from an ethical point of view, forscientific feedback on the cloning technique. Itwould be important to be able to give, thanks togenomic tools, the ‘raison d’etre’ — the object(why) and goal (purpose) of what is done butalso of what is created, since we must rememberthat we are talking about animals [3].

2. The second question is a question of animalethics: whilst the theoretical problems of cloningand transgenesis on animals are still not solved,they infer practical problems which are the sym-bols of a technically and scientifically fuzzycontext. The ethical question is, therefore: arecloned and transgenic animals supposed to bene-fit from a special ethical status, considering theiroutstanding symbolic and scientific status?

3. The third question is about research ethics con-cerning the use of mammals. Scientists find itdifficult to define and justify what they are cre-ating because of the absence of a clear definitionof the status of these ‘new beings’ (which havea concrete existence) and because of their rela-tionship with those animals [8]. We may thinkthat the responsibility for thinking of the long-term utility is required for scientists during theconstruction of new projects. Helping to definemore precisely the identity of clones would con-stitute a first step towards this fulfilling thisresponsibility.

As a conclusion, we could say that ethical approa-ches should not be reduced solely to a ‘utilitaris-tic approach’, i.e. a cost vs. benefit ratio, and


30 B. de Montera

should take into account possible help coming fromthe scientific area. Most of all, we insist on theresponsibility of the scientific community towardsthe public. Scientists should not decide on theirown in matters, e.g. life issues, natural vs. engi-neered animals, that concern the majority of humanbeings. The paradox of the present matter is thatthe resurgence of questioning about the relationshipbetween humans and animals emerges from thearea of engineered life itself. A genomic approachused in order to improve philosophical reflectionseems to be a good way to illustrate the linkbetween genomics and bioethics.

Acknowledgements

I would like to thank Jean-Paul Renard for his veryconstructive help during the preparation of the manuscript.I would also like to thank Magali Leroy and Martin Granafor their help during the re-reading.

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16. Van Reenen CG, Meuwissen TH, Hopster H, et al. 2001.Transgenesis may affect farm animal welfare: a case forsystematic risk assessment. J Anim Sci 79(7): 1763–1779.

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genomics and ethics: the case of cloned and/or transgenic animals · genomics and ethics:...

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