Somatic Cloning of ω-3 Fatty Acid Desaturase Gene Transgenic Pigs

Abstract: Pork is the most important animal foodstuff for human being, most of the polyunsaturated fatty acids (PUFAs) in pork areω-6 PUFAs, but there is quite a littleω-3 PUFAs which is healthy for us. In this study,ω-3 fatty acid desaturase gene transgenic cloned pigs were produced by using somatic cell nuclear transfer (SCNT). The results are as fallows:Experiment 1: Seven fetal fibroblast cell lines were set up from 7 fetuses of purebred England large white pigs at 40 d of gestation by using explant-seeding method. Cells after 2 d of contact inhibition were synchronized by flow cytometry and 80% cells were in G0/G1 stage.Experiment 2: Large White fetal fibroblasts were transfected with sFat-1 gene that come from round-worm C. Briggsae by lipofectamine mediated transfection, the vector is pCAGGS-sFat1-Neo expression vector, and optimized the condition of transfection and selection. Through the analysis of large white fetal fibroblast cell’s sensitivity to G418, we found 600μg/mL G418 could kill all the cells in 7 d, so the suitable condition of selection is this. When the proportion of liposome and the DNA was 10μL:3μg、10μL:4μg、10μL:6μg and 10μL:7μg, 10μL:4μg was determined to be the most effective way for transfection.18 better cell clones after the G418 were analyzed by PCR and RT-PCR, the PCR result showed that No.18、3、6、12、16 and 17 cell clones were negative; Then the rest of 12 cell clones were analyzed by RT-PCR for their RNA express level, No. 4 clone’s RNA expression was very low, we concluded it negative, the else is positive.Experiment 3: The follicles were divided into 2 groups: the large one (diameter 4-6 mm) and small one (diameter 2-3 mm). The oocytes after IVM for 38 to 42 h were used as receptor, and the percentage of cleavage and blastulast were used as test criterion to evaluate the quality of oocytes. In assistant activation, the comparison of electrical assistant activation and CHX chemistry assistant activation were investigated. The result indicated that : after 40 h IVM, oocytes from large follicles had the highest blastulast rate(19.3±1.7%,P < 0.05), small follicles’oocytes were followed(11.0±1.3%). The rate of small follicles’blastulast can be sublimated by using CHX chemistry assistant activation, but group of large follicles was not. Experiment 4: Transgenic embryos were reconstructed by nuclear transfer of positive cells into enucleated in vitro matured oocytes. A total of 1889 reconstructed embryos were transferred into 10 naturally cycling gilts. Nine early pregnancies were established, and 7 of them went to term. Twenty one piglets were born. The cloning efficiency is 1.1% (born piglets/transferred embryos). The integration of sFat-1 gene was confirmed in fifteen live cloned piglets by PCR and Southern blot except for two piglets. Expression of sFat-1 gene in 12 of 13 piglets could be detected with RT-PCR.These results indicate that SCNT could produced transgenic pigs effectively, and by cell transfection、selection and identification, higher transgenic efficiency will be achieved. The born of sFat-1 transgenic cloned pigs has established a substantial foundation for the improvement of pork quality…
Key words: somatic cell nuclear transfer; transgenic pig; ω-3 fatty acid desaturase gene sFat-1; fetal fibroblast

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