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GENETICALLY MODIFIED MAMMALS PRODUCE HEART-HEALTHY FATS
Both omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFA) are essential lipids for good health, but they are metabolically and functionally distinct and often have important opposing physiological functions1. Their balance is important for homeostasis and normal development. During the period when our genetic patterns were established, the ratio of n-6/n-3 PUFA in the diet was around 12. Deviation began ~10,000—15,000 years ago (too short a time to affect genetic adaptation significantly) with adoption of agriculture and animal husbandry, mainly of ruminants. Modern agriculture, with its dependence on grain diets, led to an increase in total saturated fatty acids and in the n-6 polyunsaturated fatty acids, linoleic and arachidonic acids. In the past century, the industrial revolution, with the emergence of agribusiness with processed foods, grain fattened livestock, and hydrogenation of vegetable fats, has further reduced the content of n-3 fatty acids and increased n-6 fatty acids (the ratio of n-6 to n-3 essential fatty acids in today's Western diets is around 15—20:1)2-3. Recent studies suggest that excessive amounts of n-6 PUFA and a very high n-6/n-3 ratio promotes the pathogenesis of many modern diseases (e.g., heart disease, cancer, etc.), while balancing or reducing the ratio of n-6/n-3 fatty acids may decrease the risk of these diseases1. Thus, for good health it is necessary to have a balance of n-6/n-3 fatty acids in the diet and in our bodies. To overcome this modern nutritional problem, there is a need to test if return of a missing fat-1 gene encoding an enzyme found in C. elegans4-5, n-3 fatty acid desaturase, which catalyzes conversion of n-6 to n-3 fatty acids, to mammals enables them to produce n-3 from n-6 fatty acids. To heterologously express the C. elegans n-3 fatty acid desaturase in mice, the fat-1 gene encoding this protein was modified from the original by optimization of codon usage for mammalian cells and coupled to a chicken beta-actin promoter and cytomegalovirus enhancer, which are highly active in a wide range of cell types and therefore allow high-level and broad expression of the transgene in mice6. The expression of the fat-1 in F1 pups from transgenic founder mice and their offspring was examined by Real-Time PCR of tail DNA and by analysis of tail lipids. The transgenic mice appear to be normal and healthy. Both transgenic and wild type mice are maintained on a diet high in omega-6 fatty acids (mainly linoleic acid) with very little omega-3 fatty acids (~0.1% of total fat supplied). Feeding this n-3 fatty acid-deficient diet allows us to identify the phenotype readily. Under this dietary regime, wild type mice have little or no n-3 fatty acid in their tissues because the animals naturally cannot produce n-3 from n-6 fatty acids, whereas the fat-1 transgenic mice should have appreciable amounts of n-3 fatty acids (derived from n-6 fatty acids) in their tissues if the transgene is functional in vivo7. Since the phenotype of the transgenic mouse lines is mainly reflected by lipid profiles, we analyzed the fatty acid composition of various organs of the transgenic mice at different ages by gas chromatography—mass spectrometer. Figure 1 shows the differential fatty acid profiles of total lipids extracted from skeletal muscles of age- and sex-matched wild type and transgenic mice. In the wild type animals, the polyunsaturated fatty acids found in the tissues are mainly (98%) n-6 linoleic acid (LA, 18:n-6) and arachidonic acid (AA, 20:4n-6) with a trace (or undetectable) amount of n-3 fatty acids.
In contrast, there are large amounts of n-3 polyunsaturated fatty acids, including linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), in the tissues of transgenic mice. Accordingly, the levels of the n-6 fatty acids LA and AA in the transgenic tissues are significantly reduced, indicating a conversion of n-6 to n-3 fatty acids. The resulting ratio of n-6 to n-3 fatty acids in the tissues of transgenic animals is close to 1. This n-3 rich lipid profile with a balanced ratio of n-6 to n-3 and an even more balanced AA/(EPA+DPA+DHA) ratio can be observed in all the organs/tissues, including muscle and milk. The muscle of the transgenic animals has the most significant change in these ratios, indicating the highest enzyme activity in this tissue. To date, four generations (homozygotes or heterozygotes) of transgenic mouse lines have been examined and their tissue fatty acid profiles show consistently high levels of n-3 fatty acids, indicating the transgene is functionally active in vivo and transmittable. Our data clearly show that the transgenic mice expressing the fat-1 gene are capable of producing n-3 fatty acids from n-6 fatty acids, resulting in enrichment of n-3 fatty acids in their organs/tissues without the need of dietary n-3 supply, which is impossible in wild type mammals7. The available sources of n-3 fatty acids in our diets are from marine vertebrates, but stem from the ability of single cell phytoplankton and algae to convert the parent n-6 fatty acid, linoleic acid, to the parent n-3 fatty acid, α-linolenic acid, which enters the food chain of marine life and is further elongated and desaturated to produce the fish oil fatty acids EPA and DHA. As sources of edible fish in the oceans are being depleted by over-fishing and the market price of fish keeps rising, the continued supply of dietary n-3 PUFAs in the future is a concern. Our findings provide a new strategy for producing n-3 PUFA-enriched foodstuff (e.g., meat, milk, and eggs) by generating large transgenic animals (e.g., cow, pig, sheep, and chicken) with the n-3 desaturase gene. In recognition of the health benefits of omega-3 fatty acids and the deficiency of these fatty acids in Western diets, a great effort has now been made to return omega-3 fatty acids to the food supply3. Since most animals cannot produce omega-3 fatty acids themselves, what the food industry is currently doing in order to enrich animal food products with n-3 fatty acids is to feed animals flax seed, fish meal, or other marine products. This feeding procedure is not only time consuming and costly, but source-limited. Thus, the feeding strategy seems to be an unsustainable method of producing omega-3 rich foodstuff. With the use of our gene transfer strategy, transgenic animals genetically capable of producing n-3 fatty acids themselves can be created. We could thus achieve an n-6/n-3 ratio approximating 1.0 by consuming foods with such a ratio without the public having to make stringent changes in their diets. In addition, availability of the "Omega-3" mice will provide a unique model and new opportunities for elucidation of the biological functions of the n-3 fatty acids as well as the importance of supplementation with these fatty acids and the balance of n-6/n-3 ratio in disease-prevention and treatment. As interest in n-3 fatty acids is growing, the significance and impact of the availability of the unique transgenic mice for n-3 research are obvious. References:1. Simopoulos AP and Cleland LG. (eds) (2003) Omega-6/omega-3 essential fatty acids ratio: The scientific evidence. World Rev. Nutr. Diet. (Basel, Karger) vol. 92. 2. Leaf A and Weber PC. (1987) A new era for science in nutrition. Am. J. Clin. Nutr. 45: 1048-1053. 3. Simopoulos AP et al. (eds) (1998) The Return of Omega-3 Fatty Acids into the Food Supply: Land-based Animal Food Products and Their Health Effects. World Rev. Nutr. Diet. (Basel, Karger) vol. 83. 4. Spychalla JP, Kinney AJ and Browse J. (1997) Identification of an animal omega-3 fatty acid desaturase by heterologous expression in Arabidopsis. Proc. Natl. Acad. Sci. USA. 94: 1142-1147. 5. Kang ZB, Ge Y, Chen ZH, Brown J, Laposata M, Leaf A and Kang JX. (2001) Adenoviral gene transfer of C. elegans n-3 fatty acid desaturase optimizes fatty acid composition in mammalian cells. Proc. Natl. Acad. Sci. USA 98: 4050-4054. 6. Niwa H, Yamamura K, and Miyazaki J. (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108: 193-199. 7. Kang JX, Wang J, Wu L, Kang ZB. (2004) Fat-1 transgenic mice convert n-6 to n-3 fatty acids. Nature 427: 504. Jing X. Kang
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