Study On Fluorescent Protein Transgenic Pigs By Somatic Cell Nuclear Transfer And Chimeric Technique

Pigs are approximately the same size as humans, their metabolism and the structure of their organs are similar; the pig genome is much closer to that of humans than the mouse genome. So pigs are regarded as primary candidates for human disease model. Pigs carrying fluorescent protein gene have an important application in xenotransplantation, chimeras and medical fields. In this study, we have obtained transgene pigs expressing GFP fluorescent protein and explored how to obtain chimeric pigs by transgenic technology,somatic cell nuclear transfer and chimeric technology. The results were as follows:1?Build Alpha-1,3galactosyltransferase gene-knockout (GTKO) miniature pigs'(BM?WZPF) ears primary fibroblast cells, using liposomes and electric transfection techniques established cell lines were transfected, G418 selection, obtain huKO, EGFP-positive cell.2?The selected positive cells was used for somatic cell nuclear transfer and embryos transfer.559 embryo were transferred to surrogates 3,1 surrogates successly deliver litter 2 piglets. After sampling, by immunohistochemistry, Western and real-time quantitative PCR analysis identified a green fluorescent protein in heart, liver, spleen, lung, kidney, pancreas, intestine, stomach, tongue, brain, cerebellum, spinal cord and skeletal muscle and other 13 kinds of tissues expression; The results showed that GFP are expressed in all tissues and organs of GTKO pig, but expressed in the liver, brain, cerebellum and spinal cord weaker.3?Microinjection iPS cell construct chimeric embryos, embryo transfer in vivo validation of pluripotency of iPS cells. Totaling 602 transplanted chimeric embryos, fetuses obtain nine. Through small animal in vivo fluorescence and Stereo fluorescence microscope observation not found EGFP expression, followed by a PCR assay,1#,2# fetal head and fetal placental EGFP gene was detected.4?Through injection and polymerization studies chimeric pigs were transplanted 436 embryos; but did not get chimeric fetus.In conclution, we had successfully developed the GFP positive GTKO pig with generalized expression by somatic cell nuclear transfer technology, a platform for production of chimeric pigs has been established.