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.

e. embryonic stem cell-mediated gene transfer and retrovirus-mediated gene transfer.. X-chromosome or Y-chromosome integration events do occur and obviously may be influenced by the choice of pronucleus. Subsequent reports (Brinster et al. Although the recombinant viral construct was proven to have integrated into the mouse genome.or under-expression of certain genes or to the expression of genes entirely new to the animal species. If this transferred genetic material (i. The transgene may be microinje cted into either of these pronuclei with equivalent results. the male and female pronuclei are microscopically visible as individual structures. It is one of the first methods that proved to be effective in mammals (Gordon and Ruddle... 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.e. into the pronucleus of a fertilized ovum. the animal will be born with a copy of this new information in every cell. the majority examining the effects of microinjected viral sequences on mammalian growth and pathology. their small size and low cost of housing in comparison to that for larger vertebrates. the pronuclear period immediately following fertilization. The introduced DNA may lead to the over. Usually. and their fairly well defined genetics. The first visible phenotypic change in transgenic mice was described in 1982 for animals expressing the rat growth hormone sequence (Palmiter et al. 1980). The three principal methods used for the creation of transgenic animals are DNA microinjection. 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. or foster mother that has been induced to act as a recipient by mating with a vasectomized male. the male pronucleus may be distinguished because it is larger than the female nucleus and also because it is closer to the oocyte surface. 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. Currently. The insertion of DNA is. several hundred transgenic expression papers are published each year. The first successful production of transgenic mice using pronuclear microinjection was reported in 1980 (Gordon et al. 1982). For several hours following the entry of the sperm into the oocyte. their short generation time. however. a random process. i. transgene) is integrated into one of the embryonic chromosomes. it was rearranged and did not express.e. For this reason. the transgene DNA is introduced into the zygote at the earliest possible stage... mice have become the main species used in the field of transgenics. However. 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. 1981. The manipulated fertilized ovum is transferred into the oviduct of a recipient female. a) DNA microinjection. . 1981) proved that integrated transgenes were capable of functional expression following pronuclear microinjection. i..Methods of creation of transgenic animals For practical reasons. 1981). Costantini and Lacy.

the genotype of the founder is described as hemizygous for the transgene rather than heterozygous.. then all descendants of this animal are members of a unique transgenic lineage. 1985.The animal that develops after receiving the transgene DNA is referred to as the founder (Fo) of a new transgenic lineage. These mutations are distinguished from the true transgenic phenotype because only a single lineage exhibits the defect. and rarely appears to be correlated with the degree of transgene expression in the animal. Many studies have found dramatic differences in the expression of a specific transgene within individual sibling embryos simply due to different integration loci. Integration of foreign DNA into the embryonic genome generally is a random event with respect to the chromosomal locus. may be produced by the mating of a pair of hemizygous F1 siblings. Because the new transgenic locus is present in only one member of a particular paired chromosome. cardiovascular. observable insertional mutagenesis might be apparent when the insertion interferes with the expression of an endogenous developmentally active gene.. in which a pair of transgene alleles is present. Therefore the probability of identical integration events in two embryos receiving the same transgene is overwhelmingly unlikely. The num ber of copies of the transgene that have joined the founder's genome is referred to as the copy number. The mutations can involve any system including the special senses. Interruption of the normal expression of an endogenous gene may be inconsequential or lethal. Bishop and Smith. 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. . 1989). 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. Because the locus of transgene integration is random. 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. the techniques of microinjection and embryo transfer to a suitable recipient female must be mastered. and severe morphogenetic abnormalities may be observed (Woychik et al. Of course. 1981. Alternatively. the transgene frequently inserts into functional genetic sequences. The identification of the locus of transgene insertion is of great value because it maps the locus of an important endogenous gene. Obviously. In addition. In addition. A homozygous genotype. 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. neurological and reproductive systems. 1985). If the germ cells of the founder (mosaic or not) transmit the transgene stably.

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

the socalled knock-out method. ES cell-mediated gene transfer is the method of choice for gene inactivation.. This technique works particularly well in mice. build molecules of DNA containing • • • the structural gene you desire (e.b) Embryonic stem cell-mediated gene transfer. These cells are then incorporated into an embryo at the blastocyst stage of development. It has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination METHOD: 1. This technique is of particular importance for the study of the genetic control of developmental processes. This method involves prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. The result is a chimeric animal. 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. Make your DNA Using recombinant DNA methods.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. Mating these will found the transgenic strain. 6. Embryo transfer • • • Prepare a pseudopregnant mouse (by mating a female mouse with a vasectomized male). and they will be heterozygous for the gene. 3. Hope that they implant successfully and develop into healthy pups (no more than one-third will). No more than 10–20% will have it. Establish a transgenic strain • • Mate two heterozygous mice and screen their offspring for the 1:4 that will be homozygous for the transgene. Select for successfully transformed cells. 5. Transfer the embryos into her uterus. 4. Inject these cells into the inner cell mass (ICM) of mouse blastocysts. The stimulus of mating elicits the hormonal changes needed to make her uterus receptive. Test her offspring • Remove a small piece of tissue from the tail and examine its DNA for the desired gene. Transform ES cells in culture Expose the cultured cells to the DNA so that some will incorporate it. 7.

Retroviruses are commonly used as vectors to transfer genetic material into the cell. To increase the probability of expression. not all cells carry the retrovirus. cell fusion techniques. mono and polyclonal antibody technology and biological processes for commercial production have altered traditional distinctions and methods (US Congress. When the transgene has integrated into the germ cells. to change the characteristics of plants or animals. generally a virus or a plasmid. Office of Technology Assessment. 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. The novel uses of biological techniques such as recombinant DNA techniques. At this stage embryos carrying the transgene can be frozen and stored for subsequent implantation. Genetic manipulations at the level of DNA have also changed long held views as to what is considered to be animal. 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. For any of these techniques the success rate in terms of live birth of animals containing the transgene is extremely low. taking advantage of their ability to infect host cells in this way. Providing that the genetic manipulation does not lead to abortion. Transgenic Animals as Biotechnology Transgenic animals are just one in a series of developments in the area of biotechnology. gene transfer is mediated by means of a carrier or vector. the result is a first generation (F1) of animals that need to be tested for the expression of the transgene. or to develop micro-organisms for specific uses. Offspring derived from this method are chimeric. 1989).e. 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. plant and human. resulting in mice carrying this DNA. Transmission of the transgene is possible only if the retrovirus integrates into some of the germ cells. Depending on the technique used..c) Retrovirus-mediated gene transfer. 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. resulting in a chimera. i. This method involves:26 • • retroviruses used as vectors to transfer genetic material into the host cell. the F1 generation may result in chimeras. A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA. . These terms now include the use of living organisms or their parts to make or modify products. 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. In turn. not forgetting that this impacts on wider considerations of human responsibility for the welfare of other life forms. Biotechnology has transformed the way in which we understand processes such as engineering and manufacturing.

A representative. a reduction in the number of animals used. 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. so as with any other system care must be taken in drawing conclusions from the data. On the other hand. in the pharmaceutical industry. meat production. targeted production of pharmaceutical proteins. However. the use of xenografts. list of purposes for which transgenic animals have been used indicates the wide ranging application of this biotechnology: • • • • • • • • in medical research. antibodies. the success of the method has led to using its potential for investigating a wider range of diseases and conditions. In the long term. However. transgenic animals provide a means of evaluating genetic modifications in terms of anatomical and physiological changes in a complex system. at least at the public health level makes animal and human welfare inseparable. etc. is possible due to a greater specificity of the transgenic models developed.or under-expression of a modified gene (the inserted transgene). . and developing animals specially created for use in xenografting. in biotechnology: as producers of specific proteins. sheep and goats as processing units to manufacture proteins or as organ donors. but non-inclusive. genetic engineering of livestock and in aquaculture affecting modification of animal physiology and/or anatomy. for example the oncomouse with its increased susceptibility to tumor development enables results for carcinogenicity studies to be obtained within a shorter time-frame. swine. The actual use of some species may be increased. cloning procedures to reproduce specific blood lines. 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. transgenic animals are used to identify the functions of specific factors in complex homeostatic systems through over. The successful cloning of Dolly underlines the fact that innovative developments in animal science are part of the mainstream of biotechnology. This shift in the patterns of animal use is being monitored by the CCAC through the use of the Animal Use Data Form. In addition. models are not strict equivalents. Transgenic models are more precise in comparison to traditional animal models. for example to study human diseases. The complex interactive processes of living mammals are not reproducible in vitro. genetically engineered hormones to increase milk yield.The creation of transgenic animals is resulting in a shift from the use of higher order species to lower order species. in mammalian developmental genetics. and is also affecting the numbers of animals used. The potential of the technology has also made it possible to consider employing cattle. 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. drug production and product efficacy testing. in toxicology: as responsive test animals (detection of toxicants). in addition to the numbers of animals which are sacrificed as donors during the creation process. in molecular biology. thus reducing the course of tumor development in experimentally affected animals.

. In addition. or kidney. the first transgenic cow.15. In 1997. especially since residue of the hormones remained in the animal product Disease resistance Scientists are attempting to produce disease-resistant animals. Nutritional supplements and pharmaceuticals Products such as insulin. and blood anti-clotting factors may soon be or have already been obtained from the milk of transgenic cows.17 Traditional breeding is a time-consuming.4 grams per litre. increased milk production. Research is also underway to manufacture milk through transgenesis for treatment of debilitating diseases such as phenylketonuria (PKU). Xenotransplantation Patients die every year for lack of a replacement heart. difficult task. but a very limited number of genes are currently known to be responsible for resistance to diseases in farm animals. Quality Transgenic cows exist that produce more milk or milk with less lactose or cholesterol12. In the past. such as influenza-resistant pigs.APPLICATIONS: Breeding Farmers have always used selective breeding to produce animals that exhibit desired traits (e.g. it became possible to develop traits in animals in a shorter time and with more precision.000 organs are needed each year in the United Kingdom alone. pigs and cattle that have more meat on them8. or goats. high growth rate). about 5.9 Currently. produced human protein-enriched milk at 2. Rosie.. liver. When technology using molecular biology was developed. growth hormone. and cystic fibrosis. sheep. hereditary emphysema. farmers used growth hormones to spur the development of animals but this technique was problematic.25 Transgenic pigs may provide the transplant organs needed to alleviate the shortfall. 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. 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. and sheep that grow more wool18. For example. it offers the farmer an easy way to increase yields. Rosie’s milk contains the human gene alpha-lactalbumin.11.17.

Also. medical microsutures. or in which a transgene may interact with an endogenous normal or mutated gene. I. the A. For example. Therefore. By extracting polymer strands from the milk and weaving them into thread. Another use for transgenic animals involves the biological production of valuable human protein enzymes. and tennis racket strings.1 Toxicity-sensitive transgenic animals have been produced for chemical safety testing. autoimmune disease (Hammer et al... AIDS (Vogel et al. sickle cell anemia (Ryan et al.000 named genetic diseases is huge and transgenic animals could play a role. hormones and growth factors. Transgenic rodent models have been characterized for several human diseases including cardio-vascular disease (Walsh et al. Targeted production of pharmaceutical proteins. flexible material that could be used in such applications as military uniforms. the scientists can create a light. 1990). transgenic animals may be utilized to study the regulation of a specific genetic sequence in a realistic fashion.. The generation of novel cell lines from transgenic organs also promises to reduce the number of research animals required to evaluate a therapeutic compound. The phenotype and regulatory parameters of the gene then may be evaluated in an animal model with a relatively short generation time. two scientists at Nexia Biotechnologies in Canada spliced spider genes into the cells of lactating goats. 1986).. cancer (Sinn et al.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. tough. a characterized genetic sequence may be evaluated within the specific genomic background of the whole animal. Microorganisms have been engineered to produce a wide variety of proteins. transgenic genomes may be created in which more than one transgene may interact. The goats began to manufacture silk along with their milk and secrete tiny silk strands from their body by the bucketful. The use of transgenic disease models in biomedical research promises to accelerate dramatically the development of new human diagnostic and therapeutic treatments. The predictability of many transgenic phenotypes permits the i nnovative testing of diagnostic and therapeutic agents while using a reduced population of experimental animals.. normal rodent genetics and physiology are highly characterized. . 1987). 1988). Many uses have been developed and many more are forecast. 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. 1990). In 2001. The potential for treatments for the 5. Virtanen Institute in Finland produced a calf with a gene that makes the substance that promotes the growth of red cells in humans. 1990) and neurological disease (Small et al. 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. In addition. particularly in three areas: Models of human disease processes. Using transgenic techniques..

The most controversial aspect of transgenic animal usage involves the "selective improvement" of species by the modification of the genome. The goals of this type of experiment may include decreased body fat. The use of transgenic animals. but selective deletion of specific genes or regions has been attempted. At present. as bioreactors ("pharmaceutical pharming") is a cost-effective alternative to cell culture methods. Most often. Animals automatically supplement thei r bodily fluids with fresh nutrients. particularly larger mammals. increased speed. 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. lactating mammary gland or kidney. reliably regulate their internal temperature and pH and resist pathogens. remove waste products. 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.These products may be recombinant or mutated. and collection of the functional protein from the animal employs tissue-specific regulatory DNA sequences. Current techniques in the biotechnology industry use large-scale cell cultures to generate products in biological systems. 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. with the complex secretory cell types and organs of the mammalian organism. . novel disease resistance or higher yields of meat or milk. By directing (or targeting) the expression of the transgene product so that it is produced by the secretory cells of the liver. can perform much more complicated protein modifications than simply cultured cells Modification of animal anatomy and physiology. foreign genes are added to the host genome. Transgenic animals are not only cost-effective bioreactors but. "pharmers" may collect and process bodily fluids with minimal effort. This medium must be correctly buffered and must be temperature-regulated and maintained pathogen-free. these types of phenotypic alterations are more realistically achieved in plants and bacteria than in animals. a strategy described below.

It is felt that by using animals for the production of pharmaceutical proteins we reduce them to mere factories. 4. constantly evolving groups. this attitude has been termed as interest-sensitive speciesism. many retroviruses have integrated into the human genome without any recognisable devaluation of our humanness. Some people feel that animals should be regarded as equal to humans in that they have the same basic rights as human beings. However. Opinion surveys in USA. This seems not to recognise that animals also are living beings which feel pleasure and pain just as we do. This is certainly a good turn of events since transgenic technology holds great potential in many fields. and vice versa. which may hamper the free exchange of scientific research? 1. Finally. medicine. 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. Conclusion Interestingly. Japan and New Zealand reveal that only 42. and comparable genes do occur in animals. and industry. respectively. But most people seem to accept some animal suffering to serve the basic interest and welfare of mankind. 5. including agriculture.The social opinion on transgenic animal research is divided almost in the middle. The main reasons for opposition of people is as follows. rather they are regarded as dynamic. • • • • • • 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. using live animals to alleviate animal suffering? Will transgenic animals radically change the direction of evolution. An argument attempts to focus on integrity of species in that each biological species has a right to exist as a separate identifiable entity.26 especially in the development of disease models. . of the people participating in the survey favour such research. But most of the known human genes are not unique. 54 and 58%. in most societies animals are relegated to a position several steps below that of man.ETHICAL ISSUES: Ethical Issues Related to Transgenic Animals . Use of animals in biotechnological research causes great suffering to the animals. water-tight entity. But biologists do not regard a species as a fixed. or before. 2. 3. may be seen by many as clouding the definition of "humanness". which may result in drastic consequences for nature and humans alike? Should patents be allowed on transgenic animals. In addition. the introduction of human genes into animals.

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