Proc. Assoc. Advmt. Anim. Breed. Genet. Vol12
IMPLICATIONS FOR PRODUCTS AND BREEDING PROGRAMS FROM DEVELOPMENTS IN GENETIC ENGINEERING
C. Moran
Department of Animal Science, University of Sydney, NSW 2006 SUMMARY
Genetic engineering has promised many benefits for animal production. Past predictions of the benefits have been somewhat overoptimistic both in the form of the benefits and the timing, but tangible benefits are beginning to flow and should increase in the near future. Genetic marker technology will provide the most benefit to animal breeders in the immediate to medium term with transgenically modified plants now ready to make substantial contributions to animal production.
Keywords: gene products, transgenics, bioreactors, gene markers INTRODUCTION
Genetic engineering has the potential to improve animal production in numerous ways, some of which are at last being realised. It can alter the nutritional properties of pastures and feeds and improve the efficiency with which animals utilise foods and produce valuable products. It can help animals to avoid disease. Finally genetic engineering can create animals capable of producing highly valuable novel products.
There are various way in which these outcomes are achieved. Many do not even involve direct genetic modifications of animals. For example, expressing the sunflower albumin 8 (SFAS) gene in the leaves of pasture plants like subclover or the seeds of crops, like lupins, can dramatically improve their nutritional properties as the SFA8 protein is relatively resistant to ruminal degradation and is very high in sulphur amino acids. Both features help to overcome the effects of limiting availability of these amino acids on animal growth and production. Field tests are underway on these modified plants. Genetic engineering technology is revolutionising vaccine production. For example, ovine footrot vaccines specific to nine or more serotypes of Dichelobacter nodosus can be routinely and cheaply produced thanks to genetic engineering. A tick vaccine, which targets the immune response of the cow to a tick gut protein, can now be mass produced either in bacterial cells or insect cells in tissue culture and also is commercially available.
It is now possible in Australia to inject grower pigs with supplementary amounts of their own growth hormone to substantially improve food conversion efficiency and leanness. The ability to efficiently mass produce porcine growth hormone in bacteria for use with special gas powered injection guns was essential to this development.
CONSUMER AND MARKET ACCEPTANCE
While ethical and economic issues affect the application of animal biotechnology, consumer and market acceptance of a modified product will be the ultimate factors determining its production by industry. If the modified product costs more instead of less, the consumer may reject it. If there are
legitimate concerns about animal welfare or product safety, particularly residues, which can be exploited by opponents of this type of technology, it may be rejected in the market place. Even fear of unknown dangers from use or consumption of modified products can be exploited by opponents of animal biotechnology.
The major issues influencing application, regulation and acceptance of animal biotechnology can be categorised under seven headings, namely Efficacy and Economic Efficiency, Animal Welfare, Residues and Product Safety, Environmental Safety, Property Rights and Patenting, Conservation of Genetic Resources and finally Fear of the Unknown (Pandora’s Box). The thorough process of evaluation and review required for registration of recombinant porcine somatotropin, rPST (Anonymous, 1994) by the National Registration Authority illustrates just how many regulatory and advisory organisations are involved in demonstrating that a recombinant product is efftcacious and economically beneficial, as well as being safe both for consumer and worker, as well as not adversely affecting animal welfare. The provision of additional benefits for society such as a healthier human diet containing less saturated fat is also an important selling point for this product.
In this brief review, it will be impossible to cover all of these issues. However product labelling is worthy of specific mention as it may strongly influence consumer acceptance if any ethical or quality issue has been raised. Personally I support provision of information to assist informed decision making by the consumer. Thus I am not averse to labelling of products to indicate that they have been genetically engineered. Indeed this could well become an important selling point, for example the low fat content of pork from pigs treated with rPST. However the issue of special labelling is fraught with difftculties. For example, meat from pigs treated by injection with rPST does not have higher levels of PST than non-treated pigs about 24 hours after the last injection.
Should there be a legal requirement that meat from such animals be specially labelled simply because they have been treated with a recombinant product? If a sheep is fed on genetically engineered lupins, should the sheep meat be labelled as a product of genetic engineering? Finally, compulsory product labelling can create major organisational difficulties for processors dealing with both genetically engineered and non-modified products in the same stream of raw materials, as there are major practical difficulties of keeping track of the modified products.
BIOREACTORS AND XENOTRANSPLANTATION
Historically farm animals have been the source of many medically important products. For example, porcine and bovine insulin have been and still are used for the treatment of diabetes, replacement heart valves are obtained from pigs, and oxytocin from animals is used for induction of childbirth. Transgenic technology promises to extend and improve the scope for the production of medically important products from farm animals, whether pharmacologically important human proteins or modified animal organs for transplantation. While many proteins can be expressed in microbial fermentation systems, some proteins require special post-translational processing and modifications which only higher eukaryotic cells are capable of providing. Whole-body transgenic domestic animals can meet these requirements. The obvious organ for production of these pharmacological compounds is the mammary gland since milk can be collected painlessly and repeatedly for the collection and purification of the desired protein product, and regulatory
Proc. Assoc. Advmt. Anim. Breed. Genet. Voll2 sequences for well-characterised milk protein genes can be used to direct the expression of any human protein to the mammary gland. There are numerous cases of domestic animals becoming bioreactors for human proteins. However just one example gives an idea of the potential.
Deficiency of -l-antitrypsin (h ,AT) leads to emphysema. Treatment requires 200 grammes of protein per year, which costs about $22,OOO/patient when purified fi-om human blood. There are approximately 20,000 patients in the US alone, so the total world market for this product is currently probably worth in the vicinity of US$l billion. Transgenic sheep have been made which produce up to 37.5 g h ,AT/litre of milk with h ,AT constituting more than half of the protein of the milk. If the cost of producing h ,AT were halved as a result of this technology, such milk would still be worth more than US$2000/litre. There are numerous other examples of potential value adding to milk from domestic animals, although these opportunities are restricted to biotechnology companies.
Surprisingly, transgenically modified organs from pigs may, perhaps even within five years, begin to meet the huge demand for organs for human transplantation. It has been estimated that as few as four or five transgenic modifications of pigs would make many of their organs suitable for transplantation to humans. Most of these modifications are the addition of human genes to humanise the organs to mollify the immune response of the recipient and are technically possible already and almost certainly have been performed in several labs internationally. One vital modification is the removal of a gene from the pig, a glycosyl transferase, whose product elicits a hyperacute immune response. Specific removal of this gene function is technically feasible in mice where embryonic stem (ES) cell culture is feasible, but is held up by the need to develop ES cell culture for the pig. Again the economic possibilities are enormous but relevant to biotechnology companies rather than conventional animal producers.
GENETIC MARKERS AND GENE MAPPING
DNA technology provides markers for numerous applications relevant to animal breeding, health and marketing of animal products. Regardless of the type of application, the use of similar molecular technology, including DNA hybridisation, PCR amplification and DNA sequencing, are common. These markers fall under two major headings, monomorphic markers and polymorphic markers. Intraspecific variation is neither necessary nor desirable for the fast category as they are used to test for the presence of DNA from a particular species. They are used in testing for microbes in food quality, pathology or environmental contamination applications. As well, they test for consistency with product description, such as identifying the species of origin of a product, like meat, where substitution is suspected. By contrast, variation is essential for application of polymorphic markers and often the more variable the marker, the more informative it will be in its application. These applications include verifying or determining the identity of particular animals, parentage and pedigree testing, prenatal diagnosis of sex, diagnosis of inherited diseases and genetic mapping.
COAT COLOUR GENES
Recent breakthroughs in the molecular biology of coat colour inheritance in domestic animals have provided polymorphic DNA markers which promise economic benefits to the mainstream animal
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industries in the immediate future. In the pig industry for example, breeding of white animals is highly desirable as consumers prefer pork with unpigmented skin. Now a commercial DNA test is available which will permit the breeding of stocks to achieve this end reliably. Similarly in Merino sheep, any pigmented fibres contaminating the clip, from animals with entirely black fleece or even from black spots, attract harsh financial penalties. Thus any improvement in our understanding of the inheritance of pigmented wool will improve breeding for avoidance of this contamination.
Following extensive research on coat colour inheritance and pigmentation genes in mice and humans, two genes have been very well characterised in domestic animals. The dominant White mutation in pigs is due to mutation at a locus originally identified in humans as the c-Kit oncogene (cancer causing gene) and now identified as a receptor tyrosine kinase. Johansson Moller et al.
(1996) have shown that dominant white in pigs is due to a duplication of part or all of the KIT gene, causing a complete lack of melanocytes in the skin and hair. The Extension locus which encodes the melanocyte-stimulating hormone receptor (MSHR) also has been characterised at the molecular level in domestic animals. When melanocyte-stimulating hormone (MSH) binds to this receptor, it regulates tyrosinase activity within pigment cells, with high levels of tyrosinase resulting in eumelanin (dark colours, like brown or black), and low levels giving phaeomelanin (light colours, like red or yellow). One class of dominant mutation at this locus causes the receptor to be permanently switched on even in the absence of MSH and thus results in overproduction of dark pigment. The other class of recessive mutation renders the receptor unresponsive to MSH and thus results in light colour. Khmgland et al. (1995) have observed both types of mutation in Norwegian and Icelandic cattle. A mis-sense mutation, which changes the 99th amino acid from leucine to proline, causes the MSHR molecule to permanently signal for expression of tyrosinase, with black coat colour resulting. Alternatively a single nucleotide deletion completely destroys the ability of the gene to encode a receptor which can either respond to MSH or signal for tyrosinase expression and the result is a recessive red coat colour. Joerg et al (1996) have shown independently that red coat colour in Holstein Friesians is also caused by a deletion mutation. For horses, Marklund et al. (1996) have shown that a recessive missense mutation is responsible for the chestnut colour.
The Ago&i locus has been characterised molecularly in mice and humans. The product of this gene is the agouti signalling protein, which regulates the relative production of phaeomelanin and eumelanin, but strangely is also implicated in diabetes and obesity. Preliminary evidence (parsons et al. 1997) implies that recessive black Merinos are caused by a mutation at this locus. It will only be a matter of time before the precise molecular nature of the mutation is revealed in sheep.
Because each of these mutations has been characterised at the molecular level, simple DNA tests can identify carriers of recessive genes. These tests can be used to produce true breeding lines for dominant genes, like white pigs or white sheep, which do not have recessive pigmentation genes lurking within them. Also they can identify carriers of pigmentation genes to breed lines of pigmented animals if so desired.
Proc. Assoc. Advmt. Anim. Breed. Genet. Vol12 GENE MAPPING
Over the past ten years, genetic maps for domestic animals have grown from virtually nothing to the stage where there are numerous markers on all chromosomes in most species. The justification for the expenditure on this research effort has been identification of genes causing variation in economically important traits. Pioneering work by Andersson et al. (1994) in pigs revealed the potential of this approach with quantitative trait loci (QTL) for fatness and intestine length being recognised. Numerous other groups have taken up the challenge of identifying these important genes in cattle, both dairy and beef, pigs, sheep and other species. Hetzel et al. (1997) have reported identification of QTL for carcass and meat quality in tropical beef cattle in Australia.
Undoubtedly the identification and utilisation of genes identified in this way will become more and more important over the next few years, both for marker assisted selection (MAS) and for positional cloning and molecular exploitation of the genes involved.
CANDIDATE GENES
Because of their biochemical and physiological role, some genes are candidates for producing variation in economically important traits. DNA markers based on them are thus useful adjuncts to selection, especially for difficult to select traits, like slaughter traits or reproductive traits.
Rothschild et al. (1996) investigated the estrogen receptor locus as a candidate for litter size variation in the pig and found allelic variants associated with differences in litter size of about 1.5 piglets per litter. When QTL have been mapped, the positional candidate approach can be used to exclude loci which do not map to the same genomic region as the QTL. This positional candidate approached is greatly helped by the availability of a detailed comparative map which allows exploitation of information from other species, particularly human and mouse, which have very extensive maps and molecular data available.
CONCLUSIONS
Many DNA markers are available to assist animal breeders and producers in parentage and sex verification and in tracking the inheritance of recessive genes, and many more will become available in future. Most of these tests are commercially available and can be performed under licence. Other advantages of genetic engineering to animal production are arising from the cheap availability of proteins therapeutic or other applications in the animals. Transgenic animals as bioreactors for producing human or other proteins are a growing reality, but the modification of production animals by transgenesis to improve productivity, has not had any commercial impact anywhere in the world and remains beset by regulatory hurdles and other difftculties.
REFERENCES
Andersson, L., Haley, C.S., Ellegren, H. et al. (1994) Science 263: 177 1.
Anonymous (1994) “Porcine Somatotropin (PST)” PSTInformution Service, Drummoyne, NSW.
Hetzel, D.J.S., Davis, G.P. et al. (1997) Proc. Assoc. Advmt. Anim. Breed Genet. 12:442 Joerg, H., Fries, H.R., Meijerink, E., Stranzinger, G.F. (1996) Mammalian Genome 7: 317.
Johansson Moller, M., Chaudhary, R., Helhnen, E. et al. (1996) Mammalian Genome 7: (in press).
Khmgland, H., Vage, D.I., Gomezraya, L., et al. (1995) Mammalian Genome 6: 636.
Marklund, L., Johansson Moller, J., Sandberg, K. et al. (1996) Mammalian Genome 7: (in press)
Parsons, Y&l:., Fleet, B&R. aud. Cooper, D.W. (1997). Proc. hsoc. Advmt. Ani= Breed &net.
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Rothschild, M., Jacobson, C., Vaske, D. et al. (1996) Proc. Nat. Acad Sci. USA 93: 201
