Production Of GTKO/LEA29Y Transgenic Pigs By Using The TALEN And Porcine Insulin Promoter

Nowadays, diabetes has become a global threat of human health.Islet transplantation is an effective method to cure severe diabetes, but its development is restricted by the shortage of donated organs. Xenotransplantation is an effective way to resolve the shortage of organ donor.The successful knockout of α-1.3 galactose glucoside transferase gene GGTA1 has solved the primarily hyperacute rejection. However, T cell mediated rejection is still a obstacle restricting the development of xenotransplantation. LEA29Y is a new fusion protein could efficiently inhibits the activation of T lymphocytes, the widely expression of LEA29Y often causes the transgenic animal a low immunity and difficulty to survive. So the producing the pancreatic-specificity express LEA29Y transgenic pigs is of great significance in solving transplantation immune rejection.Using the porcine insulin promoter (PIP) and bovine growth hormone poly A (bGHpA),we constructed a vector inducing islet β-cells specificity expression of LEA29Y, also we used the homologous recombination mediated by TALENs knockout the GGTA1 gene (GTKO), in order to obtain the GTKO/LEA29Y transgenic pigs. And the results are as follows:1, Considering the different location of restriction sites may affect the promoter’s efficiency, three different expression vectors were constructed by using the porcine insulin promoter (PIP) and bovine growth hormone poly A (bGHpA).Analyzing and comparing the experiments result, we discovered that mutant the PIP splice site would cause the first intron not spliced, but induced a more effcient expreesion of the downstream gene. The express cassette can be used for target gene expressed specificity in islet beta cells.2, We Inserted thel.2 kb nucleotide sequence of LEA29Y into the expression cassette,to replace the EGFP cDNAs. In vitro transfecting MIN-6 islet β-cells and porcine ear fibroblasts, the RT-PCR and western blot results suggests that LEA29Y only expressed exclusively in beta cells as expected.The construct can be used for the subsequent cell transfection and somatic nuclear transfer, to produce the islet-specifically expressed LEA29Y transgene pigs.3, Referring to the wuzhishan miniature pig genome sequence, a TALEN plasmid to knock out GGTA1 gene was designed, and co-transfected wuzhishan miniature ear fibroblasts with the islet LEA29Y gene specific expression plasmid. After screening of cells we got desired GTKO/LEA29Y single cell clonings, the GTKO/LEA29Y embryos produced by somatic cell nuclear transfer technology was transplantation into replacement gilt to obtain the corresponding positive transgenic pigs,.