TRANSGENIC ANIMALS

INTRODUCTION:

The term transgenic animal refers to an animal in which there has been a deliberate modification of the genome - the material responsible for inherited characteristics - in contrast to spontaneous mutation (FELASA September 1992, revised February 1995). Foreign DNA is introduced into the animal, using recombinant DNA technology, and then must be transmitted through the germ line so that every cell, including germ cells, of the animal contain the same modified genetic material.

Historical background

Prior to the development of molecular genetics, the only way of studying the regulation and function of mammalian genes was through the observation of inherited characteristics or spontaneous mutations. Long before Mendel and any molecular genetic knowledge, selective breeding was a common practice among farmers for the enhancement of chosen traits, e.g., increased milk production.

During the 1970s, the first chimeric mice were produced (Brinster, 1974). The cells of two different embryos of different strains were combined together at an early stage of development (eight cells) to form a single embryo that subsequently developed into a chimeric adult, exhibiting characteristics of each strain.

The mutual contributions of developmental biology and genetic engineering permitted rapid development of the techniques for the creation of transgenic animals. DNA microinjection, the first technique to prove successful in mammals, was first applied to mice (Gordon and Ruddle, 1981) and then to various other species such as rats, rabbits, sheep, pigs, birds, and fish. Two other main techniques were then developed: those of retrovirus-mediated transgenesis (Jaenisch, 1976) and embryonic stem (ES) cell-mediated gene transfer (Gossler et al., 1986).

Since 1981, when the term transgenic was first used by J.W. Gordon and F.H. Ruddle (1981), there has been rapid development in the use of genetically engineered animals as investigators have found an increasing number of applications for the technology.

Methods of creation of transgenic animals

For practical reasons, i.e., their small size and low cost of housing in comparison to that for larger vertebrates, their short generation time, and their fairly well defined genetics, mice have become the main species used in the field of transgenics.

The three principal methods used for the creation of transgenic animals are DNA microinjection, embryonic stem cell-mediated gene transfer and retrovirus-mediated gene transfer.

a) DNA microinjection.

This method involves the direct microinjection of a chosen gene construct (a single gene or a combination of genes) from another member of the same species or from a different species, into the pronucleus of a fertilized ovum. It is one of the first methods that proved to be effective in mammals (Gordon and Ruddle, 1981). The introduced DNA may lead to the over- or under-expression of certain genes or to the expression of genes entirely new to the animal species. The insertion of DNA is, however, a random process, and there is a high probability that the introduced gene will not insert itself into a site on the host DNA that will permit its expression. The manipulated fertilized ovum is transferred into the oviduct of a recipient female, or foster mother that has been induced to act as a recipient by mating with a vasectomized male.

The first successful production of transgenic mice using pronuclear microinjection was reported in 1980 (Gordon et al., 1980). Although the recombinant viral construct was proven to have integrated into the mouse genome, it was rearranged and did not express. Subsequent reports (Brinster et al., 1981; Costantini and Lacy, 1981) proved that integrated transgenes were capable of functional expression following pronuclear microinjection. The first visible phenotypic change in transgenic mice was described in 1982 for animals expressing the rat growth hormone sequence (Palmiter et al., 1982). Currently, several hundred transgenic expression papers are published each year, the majority examining the effects of microinjected viral sequences on mammalian growth and pathology.

The pronuclear microinjection method of producing a transgenic animal results in the introduction of a purified double-stranded DNA sequence into the chromosomes of the fertilized mammalian egg. If this transferred genetic material (i.e., transgene) is integrated into one of the embryonic chromosomes, the animal will be born with a copy of this new information in every cell. The foreign DNA must integrate into the host genome prior to the doubling of genetic material that precedes the first cleavage or a mosaic animal may be produced in which many cells do not possess the new gene. For this reason, the transgene DNA is introduced into the zygote at the earliest possible stage, i.e., the pronuclear period immediately following fertilization. For several hours following the entry of the sperm into the oocyte, the male and female pronuclei are microscopically visible as individual structures. The transgene may be microinje cted into either of these pronuclei with equivalent results. However, X-chromosome or Y-chromosome integration events do occur and obviously may be influenced by the choice of pronucleus. Usually, the male pronucleus may be distinguished because it is larger than the female nucleus and also because it is closer to the oocyte surface.

The animal that develops after receiving the transgene DNA is referred to as the founder (Fo) of a new transgenic lineage. If the germ cells of the founder (mosaic or not) transmit the transgene stably, then all descendants of this animal are members of a unique transgenic lineage. Integration of foreign DNA into the embryonic genome generally is a random event with respect to the chromosomal locus. Therefore the probability of identical integration events in two embryos receiving the same transgene is overwhelmingly unlikely. In addition, it is impossible to regulate exactly how many copies of the transgene will be introduced into the embryo and how many will join together to integrate (usually at a single site) as a single linear array called a concatamer (Brinster et al., 1981, 1985; Bishop and Smith, 1989). Many studies have found dramatic differences in the expression of a specific transgene within individual sibling embryos simply due to different integration loci. The num ber of copies of the transgene that have joined the founder's genome is referred to as the copy number, and rarely appears to be correlated with the degree of transgene expression in the animal.

Because the locus of transgene integration is random, the transgene frequently inserts into functional genetic sequences. Interruption of the normal expression of an endogenous gene may be inconsequential or lethal. Alternatively, observable insertional mutagenesis might be apparent when the insertion interferes with the expression of an endogenous developmentally active gene. These mutations are distinguished from the true transgenic phenotype because only a single lineage exhibits the defect. The mutations can involve any system including the special senses, cardiovascular, neurological and reproductive systems, and severe morphogenetic abnormalities may be observed (Woychik et al., 1985). The identification of the locus of transgene insertion is of great value because it maps the locus of an important endogenous gene.

Because the new transgenic locus is present in only one member of a particular paired chromosome, the genotype of the founder is described as hemizygous for the transgene rather than heterozygous. A homozygous genotype, in which a pair of transgene alleles is present, may be produced by the mating of a pair of hemizygous F1 siblings. Obviously, mating a pair of animals with identical transgenes but from different founder lineages cannot result in a true homozygote in which independe nt segregation of the loci is predictable.

The success of the microinjection technique relies upon the careful collection of a relatively large group of accurately timed embryos from a reproductively synchronized group of female embryo donors. In addition, the techniques of microinjection and embryo transfer to a suitable recipient female must be mastered. Of course, the combined success of all of these manipulative skills ultimately depends upon the fastidious construction and preparation of the transgene DNA fragments to be injected.

A major advantage of this method is its applicability to a wide variety of species.

b) Embryonic stem cell-mediated gene transfer.

This method involves prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. Stem cells are undifferentiated cells that have the potential to differentiate into any type of cell (somatic and germ cells) and therefore to give rise to a complete organism. These cells are then incorporated into an embryo at the blastocyst stage of development. The result is a chimeric animal. ES cell-mediated gene transfer is the method of choice for gene inactivation, the so-called knock-out method.

This technique is of particular importance for the study of the genetic control of developmental processes. This technique works particularly well in mice. It has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination

METHOD:

1. Make your DNA

Using recombinant DNA methods, build molecules of DNA containing the structural gene you desire (e.g., the insulin gene) vector DNA to enable the molecules to be inserted into host DNA molecules

promoter and enhancer sequences to enable the gene to be expressed by host cells

2. Transform ES cells in culture

Expose the cultured cells to the DNA so that some will incorporate it.

3. Select for successfully transformed cells.

4. Inject these cells into the inner cell mass (ICM) of mouse blastocysts.

5. Embryo transfer

Prepare a pseudopregnant mouse (by mating a female mouse with a vasectomized male). The stimulus of mating elicits the hormonal changes needed to make her uterus receptive.

Transfer the embryos into her uterus.

Hope that they implant successfully and develop into healthy pups (no more than one-third will).

6. Test her offspring

Remove a small piece of tissue from the tail and examine its DNA for the desired gene. No more than 10–20% will have it, and they will be heterozygous for the gene.

7. Establish a transgenic strain

Mate two heterozygous mice and screen their offspring for the 1:4 that will be homozygous for the transgene.

Mating these will found the transgenic strain.

c) Retrovirus-mediated gene transfer.

A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA. This method involves:26

retroviruses used as vectors to transfer genetic material into the host cell, resulting in a chimera, an organism consisting of tissues or parts of diverse genetic constitution

chimeras are inbred for as many as 20 generations until homozygous (carrying the desired transgene in every cell) transgenic offspring are born

The method was successfully used in 1974 when a simian virus was inserted into mice embryos, resulting in mice carrying this DNA.

To increase the probability of expression, gene transfer is mediated by means of a carrier or vector, generally a virus or a plasmid. Retroviruses are commonly used as vectors to transfer genetic material into the cell, taking advantage of their ability to infect host cells in this way. Offspring derived from this method are chimeric, i.e., not all cells carry the retrovirus. Transmission of the transgene is possible only if the retrovirus integrates into some of the germ cells.

For any of these techniques the success rate in terms of live birth of animals containing the transgene is extremely low. Providing that the genetic manipulation does not lead to abortion, the result is a first generation (F1) of animals that need to be tested for the expression of the transgene. Depending on the technique used, the F1 generation may result in chimeras. When the transgene has integrated into the germ cells, the so-called germ line chimeras are then inbred for 10 to 20 generations until homozygous transgenic animals are obtained and the transgene is present in every cell. At this stage embryos carrying the transgene can be frozen and stored for subsequent implantation.

Transgenic Animals as Biotechnology

Transgenic animals are just one in a series of developments in the area of biotechnology. Biotechnology has transformed the way in which we understand processes such as engineering and manufacturing. These terms now include the use of living organisms or their parts to make or modify products, to change the characteristics of plants or animals, or to develop micro-organisms for specific uses. The novel uses of biological techniques such as recombinant DNA techniques, cell fusion techniques, mono and polyclonal antibody technology and biological processes for commercial production have altered traditional distinctions and methods (US Congress, Office of Technology Assessment, 1989). Genetic manipulations at the level of DNA have also changed long held views as to what is considered to be animal, plant and human. In turn, these changes have made it more difficult to evaluate the ways in which animals are used and have obscured distinctions between pure and applied research.

Consideration of the acceptability of creating specific transgenic animal strains or genetic manipulation involving interchanging DNA between species and kingdoms could be a simple animal care issue or a societal decision. The following is an attempt to show what the ability to create transgenic animals or engage in other forms of DNA manipulation means in terms of traditional ACC functions, not forgetting that this impacts on wider considerations of human responsibility for the welfare of other life forms.

The creation of transgenic animals is resulting in a shift from the use of higher order species to lower order species, and is also affecting the numbers of animals used. This shift in the patterns of animal use is being monitored by the CCAC through the use of the Animal Use Data Form.

An example of the replacement of higher species by lower species is the possibility to develop disease models in mice rather than using dogs or non-human primates.

In the long term, a reduction in the number of animals used, for example to study human diseases, is possible due to a greater specificity of the transgenic models developed. On the other hand, the success of the method has led to using its potential for investigating a wider range of diseases and conditions. The actual use of some species may be increased, in addition to the numbers of animals which are sacrificed as donors during the creation process. The potential of the technology has also made it possible to consider employing cattle, swine, sheep and goats as processing units to manufacture proteins or as organ donors.

The complex interactive processes of living mammals are not reproducible in vitro. However, transgenic animals provide a means of evaluating genetic modifications in terms of anatomical and physiological changes in a complex system. Transgenic models are more precise in comparison to traditional animal models, for example the oncomouse with its increased susceptibility to tumor development enables results for carcinogenicity studies to be obtained within a shorter time-frame, thus reducing the course of tumor development in experimentally affected animals. However, models are not strict equivalents, so as with any other system care must be taken in drawing conclusions from the data.

A representative, but non-inclusive, list of purposes for which transgenic animals have been used indicates the wide ranging application of this biotechnology:

in medical research, transgenic animals are used to identify the functions of specific factors in complex homeostatic systems through over- or under-expression of a modified gene (the inserted transgene);

in toxicology: as responsive test animals (detection of toxicants);

in mammalian developmental genetics;

in molecular biology, the analysis of the regulation of gene expression makes use of the evaluation of a specific genetic change at the level of the whole animal;

in the pharmaceutical industry, targeted production of pharmaceutical proteins, drug production and product efficacy testing;

in biotechnology: as producers of specific proteins;

genetically engineered hormones to increase milk yield, meat production; genetic engineering of livestock and in aquaculture affecting modification of animal physiology and/or anatomy; cloning procedures to reproduce specific blood lines; and

developing animals specially created for use in xenografting.

Important general considerations include the extent to which experience acquired in the laboratory with regard to husbandry should influence industry standards for keeping animals created specifically as living machines for the production of proteins, antibodies, etc. The

successful cloning of Dolly underlines the fact that innovative developments in animal science are part of the mainstream of biotechnology. In addition, the use of xenografts, at least at the public health level makes animal and human welfare inseparable.

APPLICATIONS:

Breeding

Farmers have always used selective breeding to produce animals that exhibit desired traits (e.g., increased milk production, high growth rate).11,15,17 Traditional breeding is a time-consuming, difficult task. When technology using molecular biology was developed, it became possible to develop traits in animals in a shorter time and with more precision. In addition, it offers the farmer an easy way to increase yields.

Quality

Transgenic cows exist that produce more milk or milk with less lactose or cholesterol12, pigs and cattle that have more meat on them8,17, and sheep that grow more wool18. In the past, farmers used growth hormones to spur the development of animals but this technique was problematic, especially since residue of the hormones remained in the animal product

Disease resistance

Scientists are attempting to produce disease-resistant animals, such as influenza-resistant pigs, but a very limited number of genes are currently known to be responsible for resistance to diseases in farm animals.

Xenotransplantation

Patients die every year for lack of a replacement heart, liver, or kidney. For example, about 5,000 organs are needed each year in the United Kingdom alone.25 Transgenic pigs may provide the transplant organs needed to alleviate the shortfall.9 Currently, xenotransplantation is hampered by a pig protein that can cause donor rejection but research is underway to remove the pig protein and replace it with a human protein.

Nutritional supplements and pharmaceuticals

Products such as insulin, growth hormone, and blood anti-clotting factors may soon be or have already been obtained from the milk of transgenic cows, sheep, or goats. Research is also underway to manufacture milk through transgenesis for treatment of debilitating diseases such as phenylketonuria (PKU), hereditary emphysema, and cystic fibrosis.

In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk at 2.4 grams per litre. This transgenic milk is a more nutritionally balanced product than natural bovine milk and could be given to babies or the elderly with special nutritional or digestive needs. Rosie’s milk contains the human gene alpha-lactalbumin.

Human gene therapy

Human gene therapy involves adding a normal copy of a gene (transgene) to the genome of a person carrying defective copies of the gene. The potential for treatments for the 5,000 named genetic diseases is huge and transgenic animals could play a role. For example, the A. I. Virtanen Institute in Finland produced a calf with a gene that makes the substance that promotes the growth of red cells in humans.

In 2001, two scientists at Nexia Biotechnologies in Canada spliced spider genes into the cells of lactating goats. The goats began to manufacture silk along with their milk and secrete tiny silk strands from their body by the bucketful. By extracting polymer strands from the milk and weaving them into thread, the scientists can create a light, tough, flexible material that could be used in such applications as military uniforms, medical microsutures, and tennis racket strings.1

Toxicity-sensitive transgenic animals have been produced for chemical safety testing. Microorganisms have been engineered to produce a wide variety of proteins, which in turn can produce enzymes that can speed up industrial chemical reactions

The Value of Transgenic Animals

Transgenic animal systems combine the virtues of cell culture and congenic breeding strategies while avoiding the negative aspects of each system. Using transgenic techniques, a characterized genetic sequence may be evaluated within the specific genomic background of the whole animal. Therefore, transgenic animals may be utilized to study the regulation of a specific genetic sequence in a realistic fashion. Many uses have been developed and many more are forecast, particularly in three areas:

Models of human disease processes. Hundreds of transgenic rodent lines have been produced by introducing into the genome genetic sequences such as viral transactivating genes and activated oncogenes implicated in specific pathologies. The phenotype and regulatory parameters of the gene then may be evaluated in an animal model with a relatively short generation time. Also, normal rodent genetics and physiology are highly characterized. The predictability of many transgenic phenotypes permits the i nnovative testing of diagnostic and therapeutic agents while using a reduced population of experimental animals. The generation of novel cell lines from transgenic organs also promises to reduce the number of research animals required to evaluate a therapeutic compound. In addition, transgenic genomes may be created in which more than one transgene may interact, or in which a transgene may interact with an endogenous normal or mutated gene. The use of transgenic disease models in biomedical research promises to accelerate dramatically the development of new human diagnostic and therapeutic treatments. Transgenic rodent models have been characterized for several human diseases including cardio-vascular disease (Walsh et al., 1990), cancer (Sinn et

al., 1987), autoimmune disease (Hammer et al., 1990), AIDS (Vogel et al., 1988), sickle cell anemia (Ryan et al., 1990) and neurological disease (Small et al., 1986).

Targeted production of pharmaceutical proteins. Another use for transgenic animals involves the biological production of valuable human protein enzymes, hormones and growth factors. These products may be recombinant or mutated, and collection of the functional protein from the animal employs tissue-specific regulatory DNA sequences, a strategy described below. Current techniques in the biotechnology industry use large-scale cell cultures to generate products in biological systems. Eukaryotic cell s or bacteria which have taken up genetic expression sequences (or constructs) are cultured in nutrient medium which is continually replaced and from which the bioengineered product is refined. This medium must be correctly buffered and must be temperature-regulated and maintained pathogen-free. The use of transgenic animals, particularly larger mammals, as bioreactors ("pharmaceutical pharming") is a cost-effective alternative to cell culture methods. Animals automatically supplement thei r bodily fluids with fresh nutrients, remove waste products, reliably regulate their internal temperature and pH and resist pathogens. By directing (or targeting) the expression of the transgene product so that it is produced by the secretory cells of the liver, lactating mammary gland or kidney, "pharmers" may collect and process bodily fluids with minimal effort. The mammary gland probably is the most promising target tissue because it produces large amounts of protein in a temperature-r egulated fluid that may be collected daily in a non-invasive fashion. Transgenic animals are not only cost-effective bioreactors but, with the complex secretory cell types and organs of the mammalian organism, can perform much more complicated protein modifications than simply cultured cells

Modification of animal anatomy and physiology. The most controversial aspect of transgenic animal usage involves the "selective improvement" of species by the modification of the genome. Most often, foreign genes are added to the host genome, but selective deletion of specific genes or regions has been attempted. It has become apparent that merely adding genes for growth factors or hormones to the genome is a simplistic approach to altering the complex multigenic physiology of the mamma l. The goals of this type of experiment may include decreased body fat, increased speed, novel disease resistance or higher yields of meat or milk. At present, these types of phenotypic alterations are more realistically achieved in plants and bacteria than in animals.

ETHICAL ISSUES:

Ethical Issues Related to Transgenic Animals - The social opinion on transgenic animal research is divided almost in the middle. Opinion surveys in USA, Japan and New Zealand reveal that only 42, 54 and 58%, respectively, of the people participating in the survey favour such research. The main reasons for opposition of people is as follows.

Should there be universal protocols for transgenesis? Should such protocols demand that only the most promising research be permitted?

Is human welfare the only consideration? What about the welfare of other life forms?

Should scientists focus on in vitro (cultured in a lab) transgenic methods rather than, or before, using live animals to alleviate animal suffering?

Will transgenic animals radically change the direction of evolution, which may result in drastic consequences for nature and humans alike?

Should patents be allowed on transgenic animals, which may hamper the free exchange of scientific research?

1. Use of animals in biotechnological research causes great suffering to the animals. But most people seem to accept some animal suffering to serve the basic interest and welfare of mankind; this attitude has been termed as interest-sensitive speciesism.

2. It is felt that by using animals for the production of pharmaceutical proteins we reduce them to mere factories. This seems not to recognise that animals also are living beings which feel pleasure and pain just as we do.

3. Some people feel that animals should be regarded as equal to humans in that they have the same basic rights as human beings. However, in most societies animals are relegated to a position several steps below that of man.

4. An argument attempts to focus on integrity of species in that each biological species has a right to exist as a separate identifiable entity. But biologists do not regard a species as a fixed, water-tight entity; rather they are regarded as dynamic, constantly evolving groups.

5. Finally, the introduction of human genes into animals, and vice versa, may be seen by many as clouding the definition of "humanness". But most of the known human genes are not unique, and comparable genes do occur in animals. In addition, many retroviruses have integrated into the human genome without any recognisable devaluation of our humanness.

Conclusion

Interestingly, the creation of transgenic animals has resulted in a shift in the use of laboratory animals — from the use of higher-order species such as dogs to lower-order species such as mice — and has decreased the number of animals used in such experimentation,26 especially in the development of disease models. This is certainly a good turn of events since transgenic technology holds great potential in many fields, including agriculture, medicine, and industry.