Studies On Production Of Transgenic Porcine By Somatic Cell Nuclear Transfer

Transgenic animal research is one of the most hottest spots in biology, which can change thegenetic component and productive performance in animal according to mankind mind. Theproduction efficieny of transgenic animal by the traditional method of pronucleus microinjection isvery low (The effciency of transgene is about 3～5％.), which seriously restricted the developmentof transgenic animal research. In comparison with the traditional method, this technique of somaticcell nuclear transfer has many advantages when used in transgenic animal production, so somaticcell nuclear transfer is the most promised route for producing transgenic farm animal. Research onGFP transgenic pig can provide useful information for transgenic breeding of pig, human diseasemodel and human xenotransplantation.In this study, we screened the effects of the concentration of liposome and plasmid, exposuretime of cells to the liposome-plasmid complexes on GFP gene transfection of procine fetalfibroblasts (PFF), then got the positive cells expressing GFP stably after selected by G418,transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated invitro matured oocytes, the development of reconstructed embryos both in vitro and in vivo wereobserved, and GFP expression was checked as well.1. G418 concentration for selection is 500μg/ml, and for keep selection is 200μg/ml. Affer 30dselection, the pure PFF that stably expressed GFP was obtained.2. T'he maximal GFP transgene expression (3.78％) was achieved when PFF at 70％～90％confluence of passage0-3, exposed for 6h to the complexes of 2.0μl liposome (lipofect transfectionregent) and 0.8μg plasmid (pEGFP-C1) per well of a 24-well plate.3. In vitro blastocyst rate of GFP reconstructed embryos was 11.4％, GFP positive blastocystrate was 55.2％.4. Reconstructed transgenic embryos were transferred to 10 recipients, 5 of them werepregnant, 3 of them produced 6 cloned piglets in which 4 out of 6 are GFP transgenic piglets, andverified by both GFP protein expression and GFP DNA sequence analysis. The cloning pigletsdelivery rate was 1.0％, positive piglets delivery rate was 0.7％.The result showed that plasmid regent could transfect PFF efficicently, and the positive cellshave potentiality to support the development of pig. We built a perfect system that containedsomatic cell culture and passage, somatic transfection, positive cells selection and passage, and the production oftransgenic cloned embryos and transfer, it was a basic for further reseaches on the piggenome in further. This is the first group of transgenic cloning pigs born in China, and it is a greatprogress for Chinese transgenic cloning pig research.