To what extent should Animal cloning be Permitted?

- Maurizio Salvi, Ph.D.
International Forum for Biophilosophy, Univ. Leuven, Belgium
E-mail: Maurizio.Salvi@cec.eu.int
Eubios Journal of Asian and International Bioethics 12 (2002), 45.

Introduction

 In 1996 the birth of "Dolly', the first mammal cloned, has opened discussions among biologists and the public about the desirability of such a technology (Terragni 1999, Dijck 1998). The public has plaid a decisive role in decisions associated to cloning both in the US and Europe (Simon 1999). This is surprising when we think that cloning was not a new technology. The first experiments of nuclear transfer with amphibians (Rana pipiens and Xenoplus laevis) were performed in the United States and Britain during 1950s (Gordon & Colman 1999: 743-746) to study the irreversibility of the modification of genetic material of differentiated cells from adult animals. Nuclear transfer experiments were performed in amphibians in the 1960s, in mice in the 1970s, in sheep in the 1980s, and in monkeys in the 1990s. They have provided evidence that fully differentiated somatic cells retain all the genetic material of the early embryo, and that differentiation is almost entirely achieved by reversible changes in gene expression.

In this paper I will deal with implications arising from animal cloning. It is not my intention to minutely report the history of cloning (see Gordon & Colman 1999:743-746) or to analyse legal implications that such a technology may involve. Here I will explore is both the biological and ethical implications that animal cloning may rise if performed in animal breeding programmes.

Dolly Superstar

 "Dolly' is a well-known name among scientists. She is the first mammal to develop from a cell derived from adult tissue (1). Dolly derived from cells that had been taken from the udder of a 6-year old Finn Dorset ewe and cultured for several weeks in a laboratory. These cells were then fused with unfertilised eggs from which the genetic material had been removed. Biologists cultured 276 of these reconstructed eggs' for 6 days in temporary recipients. Twenty-nine of the eggs that appeared to have developed normally up until the blastocyst stage were implanted into surrogate Scottish Blackface ewes. Dolly's birth drafted a revolutionary way for producing engineering animals. In fact, the normal sexual reproduction (the fertilisation of an egg by a sperm and the inter-parental chromosomes fusion) was substituted with a reproductive system by which "DNA is removed from an unfertilised egg and the egg fused with a diploid cell containing a full set of paired chromosomes" (Wilmut, April 1997). Successively, the obtained eggs had been implanted in a surrogate mother, in whose womb they developed into lambs (2).

The successful experiment performed by members of the Roslin Institute offered a new technique for producing genetically modified animals. Instead of pronuclear injection, which leads to the production of 2-3% of transgenic animals, the nuclear transfer technique leads to an easier and more successful production of GM animals (3). To summarise, Dolly's birth has achieved the following scientific goals: (1) The complete genetic material from an adult mammalian cell has been used in the development of a new individual for the first time; and (2) donor cells, induced to exit the growth phase and become quiescent before being used for nuclear transfer, are more susceptible to reprogramming by the recipient egg cell and result in the normal development and birth of cloned offspring (Wilmut et al. 1997).

After the successful experiment with Dolly, several laboratories have begun to work on different applications of animal cloning. In 1997, Dr. Pennisi announced the cloning of a transgenic lamb (Polly) cloned from cells engineered with a marker gene and an undisclosed human gene. Dr. Pennisi's experiment showed that "foreign DNA can be introduced to a genome without disrupting the genetic interactions that guide a lamb's development" (Pennisi 1997). Successively biologists have also begun to clone goats, cattle (4) and monkeys (see Revel, 2000:43-59). Currently a number of experiments are taking place to check whether all animal species are clonable (Cohen 2000:4).

Why clone animals?

 According to the Roslin Institute in Edinburgh animal cloning can achieve a number of scientific goals: 1) Human therapeutic proteins; 2) Organs and tissues for transplants; 3) Nutriceuticals; 4) 'Humanised' cows milk Animal models of disease; 5) Cell therapy agents. In April 1997, Dr Wilmut has summarised the applications of animal cloning as follows:

Applications in Medicine
Human Therapeutic proteins Transgenic sheep, goats and cattle can be used as bioreactors to produce human protein in milk (for example: alpha-1-antitrypsin). Cloning could reduce the number of animals needed for creating a transgenic line
Xenotransplantation NTT could lead the production of pigs in which pigs' proteins inducing rejection are removed and replaced by their human counterparts.
Nutriceuticals NTT ought to produce transgenic cows producing human proteins
Animal models of disease Transgenic animals can be produced by using different animal species than the used ones. This extension might allow the creation of better models for testing treatments for human pathologies
Cell therapy NTT could solve immune rejection problems. We could use cells removed from a patient and successively reintroduce the patient's cells (bioengineered) in the organism
Applications in Biological Research
Ageing & Cancer NTT avoids DNA replication's mistakes _€somatic mutations- and subsequent ageing and cancer processes
Alternatives to Embryo Stem Cells Embryo stem cells have only been recovered from two specific strains of mice (and not from any livestock species). Nuclear transfer would allow gene targeting in other strains of mice and in other laboratory species such as rabbits and rats.
Cloning The ability to produce large numbers of genetically identical animals would have important benefits in experimental design. The advantages of genetic uniformity have been amply demonstrated in studies with inbred lines of mice. Nuclear transfer from cultured cells could provide alternative approach in species where repeated inbreeding is impractical or prohibitively expensive.
Gene Targeting in Farm Animals
Improving Transgenic Animal Production NTT can substitute a procedure called 'pronuclear injection'. The limits of this technique were: 2-3% of GMOs are transgenic, only a portion of transgenic animals completely express the modified exogenous DNA.
Opening new

Possibilities		 NTT opens the possibility of specific genetic manipulations that are currently impossible, such as the substitution of a single base in DNA (typical of many human genetic diseases)
These potential applications of the nuclear transfer technique show that both medical research and industrial biotechnology have an interest in animal cloning. In this paper, however, I will focus on animal cloning for breeding program purposes.

The extremely high similarity between clones' genotypes, in fact, would allow manufacturing organisms having improved strains of livestock. One of the major benefits of cloning, then, is the possibility of creating populations of genetically determined organisms responding to the needs of both the animal breeding industry and the research community.

Biological implications of cloning

 Contrary to the common perception, a clone is not a carbon copy of its ancestor. Gene plasticity, environmental factors, and neural topography structures differentiate clones from their parents. Even if clones have a high genetic similarity compared to their parents, the claim that a clone is a carbon copy of another individual is biologically false and it refers to genetic determinism (5). A number of biological factors deny the claim that clones are carbon copy of their ancestors (Revel 2000): 1) Dolly's mitochondrial DNA comes from the egg donor (usually mitochondrial DNA comes from the mother); 2) Dolly's immune system genes are not developed at the embryo stage (Dolly has a different immune system from her mother).

The above mentioned differentiation-factors are reinforced by the influence that environmental factors have towards the phenotypic expression of a genotype. "Phenotypic identity requires identity between genotypes, which cloning can ensure, and identity between environmental interactions, which it cannot ensure." (Eisemberg, 1999). A cloned DNA, thus, will express differently in the phenotype of different clones. Nevertheless, the similarity between two clones is so high as to show two identical individuals. This coincidence is only apparent, since two clones have a range of constitutive qualitative dissimilarities (Kolata 1998).

In 1997, Hubbard has explained in an editorial published in The Nation (1997) the non-coincidence of cloned individuals. He underlined that "Dolly has the same DNA (or genes) in the nucleus of her genes. But, although embryologists have a way of forgetting it, an egg is not an empty bag containing nothing but a nucleus, transplanted or not. Eggs also contain structural and metabolic equipment, including a complement of extranuclear DNA specific to that individual. The second ewe did not contribute her nucleus, but she did contribute the rest of contents of her egg. The reconstituted egg was than gestation in the uterus of yet another ewe. Dolly is, indeed, a nuclear DNA clone, but there is more to life than DNA for sheep."

Biologically speaking, animal cloning involves a number of implications (6). We may distinguish two levels of implications: implications for the cloned organism (individual specific consequences) and implications that such a technology may cause at a species-specific level. Let us start with the first kind of implications.

Scientists observed that the mortality of clones is high. Currently, 50% of clones perish during pregnancy (perinatal mortality). This abnormal perinatal mortality rate may suggest that cloned individuals have physiological weakness (7) (Cohen 1998:4). Another implication of cloning refers to sexual reproduction mechanisms. A clone develops from a cell derived from an adult tissue. It is not resulting from a chromosomal fusion (maternal / paternal chromosomes) but it derives from a cell of only one organism. As such, clones are anomalous biological individualities since their genotype is a copy of the nucleus of a donor.

In parallel, clones do not have a definite age and we cannot say what parental relation exists with their genitor. As Wilmut noted "One of the more interesting questions about Dolly concerns her age. As far as her DNA goes, is she one year old or seven? And will she age prematurely? The premature death of a number of clones seems to suggest that biologically speaking clones' biological clock runs faster than other animals (Cohen 1999).

At the species-specific level cloning leads to a number of biological implications (ecological and evolutionary implications). The first consequence of cloning would be the impossibility to foresee the long-term consequences that may arise from the use of such a technology. The affirmation of bioengineered gene pools, in a repetitive number, could interact with mechanisms of population genetics by causing the affirmation of specific allelic frequencies that may lead to epidemics or deleterious outcomes towards the Natural evolution (adaptation). We have no ideas about consequences that such a pressure on the Natural evolution may cause. As Eisemberg has said, "genetic homogeneity is compatible with adaptation to a very narrow ecologic niche; when that niche is perturbed (_c) extinction may follow" (Eisemberg 1999:472). Thus, cloning may cause an increase of pathogenic gene frequencies at a population level or the loss of adaptive capacities.

In parallel, a massive use of cloning may seriously damage biodiversity. The continuous mix of genetic data via sexual reproduction is a basic mechanism of natural evolution. The possibility of continuously recombining genetic data allows adaptive processes (Gordon 1999). Biologically speaking, the advantage of biodiversity is without controversy. The primary source of genetic variations in living beings is genetic mutation "and' cell division. The first one creates new genetic information that will be naturally selected over time. The second one reshuffles the random genetic changes created by mutations. Clearly a large-scale use of cloning may affect biodiversity and favour the consolidation of specific allelic frequencies at a population level having a negative impact towards the natural balances. Seen in this light, cloning is intrinsically hazardous.

To summarise, cloning causes a number of biological problems:

1) Clones are not the carbon copy of their parents, but they have an unnatural similarity rate with them so high as to distort the population concept. Members of a population are reproductively isolated from the others. This clause refers to species-specific similarities (genetic and phenotypic similarities) making the reproductive apparatus of species-specific individuals compatible. However, the reproduction-condition among members of a species does not involve their genetic coincidence. Cloning is biologically problematic since it may distort the species-concept.

2) Clones can reproduce and start a cascade of events by which a modified gene lineage could be artificially consolidated at a population level. Evolutionary mechanisms could be completely affected by cloning. Humans could cause the affirmation of gene pools that do not respond to adaptive mechanisms. Clearly, we have no ideas about the evolutionary response to these pressures. Subsequent mutational factors could consolidate gene frequencies (at a population level) that are different from the desired ones.

The above mentioned factors show that cloning is biologically problematic. Nevertheless, we should be aware that these implications concern also other applications of animal genetics (for example, transgenic animals or chimeras). Consequently, I claim that cloning does not posit different ethical implications than the ones of transgenic animals and chimeras. To what extent are we allowed to interact with the Natural evolution to create new-organisms responding to human needs? Are we under the obligation to consider unforeseeable long-term consequences of bioengineering Life as a "reasonable risk to pay' in the name of the human progress (or economic benefits)?

Animal Cloning

 To date, a number of research institutes perform animal cloning experiments every day all across the globe. This means that this application of animal biotechnology has been considered morally legitimate. This is because, as I have already said, one of the main benefits of animal cloning is the more rapid dissemination of genetic progress from elite herds to the commercial farmer. Thus, animal cloning constitutes an ideal tool to optimise both the cost/benefit model and the effectiveness of animal breeding programmes. Let us start to analyse the ethical legitimacy animal cloning by considering the benefits that such a technology may bring forward.

According to Wooliams "The basic benefit for the biotechnology industry of cloning will (be) _c that it will lead to a high optimisation of engineering organisms' production. (_c) Cloning could substitute artificial insemination and embryo transfer in breeding programmes. Farmers could receive cloned embryos of the most productive cows of elite herds. Biotechnology industries could create catalogues that describe the genetic merit of cloned organisms, their fertility, health and longevity (8) (Woolliams 1997)".

The first objection to the ethical legitimacy of Woolliams' hypothesis refers to the effectiveness of cloning. The mortality rate of cloning is high. (1 out 277 tries to clone Dolly.) At the present, both the percentage of implanted embryos that had a full-term development (2.5%, due to failure of genomic reprogramming or imprinting _€Revel, 2000:45) and the incidence of perinatal mortality, make this technology really inefficient. However, although this ineffectiveness seems to impede the use such a technology in human genetics (human reproductive cloning), it does not represent a significant obstacle to obstruct animal cloning. We can assume that, in a future, animal cloning will be more efficient (Gordon & Colman 2000:743-746). Or we can argument that other techniques used in animal biotechnology (pronuclear injection, for example) do involve a high mortality rate as well (in transgenic animals production 95-98% of genetically modified embryos are usually damaged and only 2-3% of the animals born are transgenic). According to this, as we accept the high mortality rate of producing transgenic animals, we should not think of this side effect of cloning as a relevant problem. Otherwise we should be in opposition to both animal cloning and transgenic animals production for safety reasons.

From a philosophical point of view, the capacity to better derive live animals from cultured cells may be considered as a pro cloning argument. In fact, as we think ethically legitimate the production of transgenic animals in both animal industry and research, we should claim the usability of animals in the fields of animal cloning as well. We have seen that animal cloning can increase the effectiveness of transgenic animals production (9). As such it might be accepted by animal rights advocates as a way to decrease both the mortality rate of animals used in research and animal suffering (clearly in the respect of the animal welfare clause). Ontological approaches to the animal rights issues (Regan's theory, for example), should lead to similar conclusions as well. If animals have an intrinsic value (or a moral status as subjects-of-a-life) the production of newborn organisms (clones) should be considered legitimate in itself. Particularly in those cases where clones inherit a healthy genotype that has not been modified to cause animal suffering (phenotypic expression of the cloned genotype). What arguments can we provide to discuss animal cloning, which differ from the ones posited to discuss the ethical implications of producing transgenic animals?

Can we say that two transgenes have a different moral status than two clones? Why? What difference would diversify the moral implications arising from the production of a transgene via pronuclear injection compared to the ones arising from animal cloning?

Cloning is only a technique! The ethical legitimacy of such a technology refers to the conceptual reasons by which biologists and regulatory bodies have justified the moral legitimacy of producing transgenic animals. This does not mean that animal cloning is ethically unproblematic. It simply means that the moral implications of animal cloning can be equated with the ones of manufacturing transgenic animals. Those philosophical theories that do not accept the instrumental use of animals for human purposes will automatically oppose animal cloning. On the contrary, those views that admitted such a use of animals will consider animal cloning as ethically legitimate.

In my opinion, the main problem of animal cloning refers to the ethical legitimacy of using animals for human purposes. And also in this case, my response to this puzzle does not differ from the one I have claimed elsewhere (Salvi 2001): animals are moral entities, but we can instrumentally use them in a restrictive way (in those cases in which the expected goals are so good to justify the use of animals, no alternatives are available and both the number and the suffering of animals is minimised). Then I do not oppose animal cloning, but I wonder whether we really need to clone animals. Industrial applications of cloning do not justify animal cloning, since already existing technique used in animal breeding programmes could be used to have improved strains of livestock. On the contrary, research applications of animal cloning having a clear and well-documented scientific goal (for example to explore the chemical process inducing specific cell differentiation of ES cells), should be allowed when no alternatives are available. This is not because animal cloning is ethically legitimate from a moral point of view, but simply because the value of the expected scientific goal is higher than the one of other industrial applications of animal breeding. Therefore it is the relevance of expected goal that reflects on the ethical implication of animal cloning and not the value attributed a priori to such a demonised technology (a value that does not exist to me).

I do know that some people will stress the relativity of the weighting system used to define the goodness of the chosen scientific goal. But I do think that people would accept that a clinical (or research) trial regulated by good clinical (or research) practice protocols and scientifically justified as the only tool to explore a scientific avenue which otherwise could not be followed, is not equivalent than an application of animal cloning performed to have a easier production of, say, goats with liver dysfunctions to produce higher quantity of 'p_t_ de fois gras'.

As I have said, we do know that cloning is a technique of molecular biology performed from long time. And we do also admit the possibility of animal experimentation for research purposes (regulated by 3Rs and animal welfare). Then, I do not see arguments to allow animal applications of other routine techniques of Natural sciences (such as the production of transgenic animals for research purposes) and reject cloning. If we follow the anthropocentric view that legitimates the first conclusion, we then should extend this claim to animal cloning as well. This means that I do disagree with the claim that both animals and human beings occupy the same position into a moral taxonomy. To me both the biological (linguistic and cognitive capacities) and the socio-cultural (human beings as moral-community makers) features of human beings justify the prevalence of human beings in a moral taxonomy and deny biocentric ethics. But this does not imply that I deny the moral status of non-human beings. It simply means that since human beings define the moral relevance of the actions they do animal cloning needs to be absolutely unavoidable and strictly regulated. And, following this line of reasoning, I do not see moral arguments to oppose to animal cloning for research purposes but I also do not see any reason to accept cloning as a technique to use in animal breeding programmes when the expected goals are oriented only to maximise and accelerate the animal breeding industry- oriented process.

Notes

1) The invention is covered by two patent applications filed by Roslin Institute (Edinburgh) with a priority date of 31st August 1995: PCT/GB96/02099, entitled Quiescent cell populations for nuclear transfer and PCT/GB96/02098 entitled Inactivated oocytes as cytoplast recipients for nuclear transfer. Roslin Institute (Edinburgh) does not expect any granted patents to issue for at least another 2-3 years.

2) The innovation of the nuclear transfer technique (NTT) was the use of unfertilised eggs and their fusion with a cell that contained the genetic endowment of only one organism.

3) "In cloning procedures generally, nuclei are extracted from cultured cells that might have come originally from an embryo, a fetus or an adult organism. The nuclei are inserted into egg cells which have had their original nucleus removed, a process called nuclear transfer. In the initial work at the Roslin Institute, the egg cells along with their transplanted nuclei were then implanted directly into a foster mother, where they developed and, in the case of Dolly, resulted in a viable offspring." (http://www.sciam.com/explorations/090297clone/beardsley.html).

4) The NTT technique is actually used in Denmark and Australia to clone cattle (Viborg Laboratories of Denmark's National Institute of Animal Science, and Monash University). The Danish team is using genetic material from dead cows. They emptied oocytes of their DNA, take adult cells from cows' ovaries, they fuse the empty oocyte with the empty cells and finally implant the obtained blastocyst in a surrogate cow. On this topic see Coghlan A., New Scientist 17, January 1998.

5) "Yet it seems clear that some of these concerns, at least, are based on false beliefs about genetic influence and the nature of the individuals that would be produced through cloning. Consider, for instance, the fear that a clone would not be an "individual" but merely a "carbon copy" of someone else -- an automaton of the sort familiar from science fiction. As many scientists have pointed out, a clone would not in fact be an identical copy, but more like a delayed identical twin. And just as identical twins are two separate people -- biologically, psychologically, morally and legally, though not genetically -- so, too, a clone would be a separate person from her non- contemporaneous twin. To think otherwise is to embrace a belief in genetic determinism -- the view that genes determine everything about us, and that environmental factors or the random events in human development are insignificant." (Wachbroit 1997)

6) A biological implication of cloning is that it has revolutionised the concept of totipotency. At present, we cannot think of germ cells as the "only' totipotent cells anymore because NTT allows somatic cells to originate a new organism. (ES cells have this capacity as well.) In the light of this, we can think of totipotency as a capacity to differentiate into nearly any cell type.

7) This mortality rate may decrease in a future when biologists will get a better understanding of what determinants cause the death of clones during pregnancy.

8) They could choose the sex of the embryo (male for beef and female for milk) and would be guaranteed a genotype of proven performance in either low or high input systems. The cloned embryo would be delivered to the farm in much the same way as semen straws are today, perhaps from breeding companies based overseas. (John Woolliams, Cloning in farm animal production, 1997, RIO)

9) The claim the cloning does involve a high mortality rate would offer a strong argument against cloning. However, new discoveries of biological sciences (the possibility to use embryonic stem cells rather than eggs _€New Scientist, 29 January 2000) can offer technical solutions for this problem.

References

Cohen P. (1999) "Dolly's mixture' New Scientist 1999, 4 September, p.5.

Cohen P. "Dolly's mixture' New Scientist 1999, 4 September, p.5.

Cohen P. (1998) "Cloning by numbers' New Scientist 19/26 December 1998:28-29.

Dijck J. (1998) Imagination of Genetics, Huondsmills London, Macmillan Press.

Eisemberg L. (1999) "Would cloned humans really be like sheep?' New England Journal of Medicine (340) 6:471-474.

Gordon J. & Colman A. (2000) "The future of cloning' Nature (402):743.

Gordon J. (1999) "Genetic enhancement in humans' Science (283): 2023-24.

Kolata G. (1998) Clone: The Road to Dolly & the Path Ahead Morrow/Avon ISBN 0-68815-6924.

Revel M. (2000) 'State-of-the-art on Research on Cloning of Whole Organisms', UNESCO IBC Proceedings of the Sixth Session, pp. 58.

Salvi M, (2001) "Transforming Animal Species: the Case of "Oncomouse', Ethics and Engineering Life (7), Boston-London: MIT, pp. 15-28.

Terragni F. (1999) Il Clone e il suo doppio, Milano, Mondadori.

Wachbroit R.,(1997) Genetic Encores: The Ethics of Human Cloning, Report for the Institute of Philosophy and Public Policy.

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