Production Transgenic Cloned Embryos Of Guangxi Bama Miniature Pig By Electroporation

China, as the primary country of pig-raising in the world. It also is abundant in resources of local inherited swine species. Nevertheless, the fine resources of local inherited swine species are not fully utilized,so we should enrich the methods of resource protection and enhance the use ratio of local species pig. Guangxi bama miniature pig is unique and endemic miniature pig breeds of china,its stable in genetic and phenotypic characteristics, the organs and physiological characteristics very close to human, so it used for surgical training, drug evaluation and human xenograft skin graft trials. Additionally, researches concerning guangxi bama miniature pig and transgenic clone not only Significantly impel the protect of local pig excellent genetic characteristics, but also provide perfect animal models for human medicine and biology. It is an ideal animal model to improve the medical domain for human biology.This study was prepared using kidney fibroblasts of new born bama pig by trypsin, We successfully established bama miniature pig kidney fibroblast cell system, plasmid of PEGFP-N1is used for transfection and optimize transfection the kidney fibroblasts. We used methods of electrical parameters to optimize screening transfection of electrical parameters, the screening transgenic fibroblasts containing genetically modified was successful select out.we used the transgenic fibroblasts as donor cells successfully constructed transgenic cloned embryos. We will provide support to further validate the ability of transgenic cloned embryos in vivo by embryo transfer, and try to build the survival of cloned transgenic pigs.Now,the main results were concluded as following:Experiment I、 employing Guangxi Bama mini pig at the age of2-3days after birth as the start materials, The fibroblasts cells are established from the kidney of newborn Guangxi bama mini pig. it is used for cloneExperiment II、 we explore the voltage how to effect on bama miniature pig kidney fibroblast survival rate.we used0,80,100,120,140,160voltage of electric shock on kidney fibroblasts, we found that cell survival rate decreased as the increase of voltage, we found about50%cell survival rate under the voltage of120.ExperimentⅢ In order to explore bama miniature pig kidney fibroblast cell electroporation conditions.the micropulser is used to electronically transfect, we used the plasmid PEGFP-N1transfect to the fibroblast of bama miniature pig. The survival rate of cells and the ratio of expressing green fluorescent are selected as criterion for comprehensive evaluation. pEGFP-N1were used to optimize the electroporation transfection system, After optimize the transfecion fibroblast conditions, and transfected cells were following suffered to resistance screening for establishing transgenic cell line, we found that the best voltage of electrotransfecton is120,Pulse duration20ms,2pulses.Experiment Ⅳ, transgenic and non-transgenic embryos at embryonic cleavage rate and blastocyst rate(8.1%vs6.7%) was not significantly different in both (P>0.05).In short, we use electricity transfection methods to build Bama miniature pig kidney fibroblasts genetically system, and we use transfected fibroblasts to constructing transgenic cloned embryos.