Objective Polyunsaturated fatty acids(PUFAs)play a vital role in human medicine and nutritional value. With the development of biotechnology, it is possible to change the variety and content of unsaturated fatty acid in pork by transgenic technology to synthesize PUFAs by mammalian itself, which contribute to the human nutrition and health.
Method By cloning FAT1 and FAT2 genes that modulated ω-3 polyunsaturated fatty acid dehydrogenase and Δ-12 fatty acid desaturase which were lack in pigs, a mutiple loci targeting vector that carrying the FAT2-FAT2 gene was constructed. This vector used the internal transcribe spacer sequences(ITS)that between the rRNA genes of pig as the target loci. To improve the efficiency of targeted integration, the positive and negative screening system of NEO and TK was introduced into the vector for double selection.EGFP reporter gene and Cre/Loxp system were added to the vector to screen out the positive clones of homologous recombination cells. The targeting vector with the FAT2-FAT2 gene was transfected into porcine kidney cell PK15 by liposome.
Result RT-PCR results showed thatFAT2-FAT2 gene expressed in porcine kidney cell PK15. The GC-MS results showed that the contents of ω-6 PUFAs and ω-3PUFAs in control group PN1(cells that transfected with PN1 plasmid), treatment group PF1(cells that transfected with FAT1 gene), treatment group PF1F2(cells that transfected with FAT2-FAT2 gene)were 7.9%, 7.03%, 3.92%, 5.64%, 7.01% and 9.43%, and the results showed significant differences. The ω-6/ω-3 ratio decreased significantly from 1.4 in the control group to 0.42-1 in the experimental group.
Conclusion FAT1-FAT2 mRNA can be expressed and produced by transfecting pig kidney cell PK15 with FAT2-FAT2double gene by constructing target vector, and the content of ω-3 PUFAs in transfected cells can be significantly increased, which lay a foundation for the subsequent production of transfected pig withFAT2-FAT2 double gene.
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