Factors Affecting Developmental Competence Of Porcine Cloned Embryos By Somatic Cell Nuclear Transfer

Cloned pigs by somatic cell have great potential as models for human disease and as human xenotransplantation. The efficiency of cloned piglets production has been very low, with less than 1% of transferred embryos surviving to term. This research focused on establishing a more efficient system for producing pigs by somatic cell nuclear transfer (SCNT). Systematical study had been carried on factors affecting on efficiency of SCNT by optimizing NT technique and culture conditions. The aim is to produce enough cloned embryos, which were transferred to surrogate gilts or sows.Somatic cell lines of a Chinese miniature swine were established: fetal fibroblast cells, ear fibroblast cells and granulosa cells. Growth curve of pig fetal fibroblast cells and synchronization treatment of cell cycle were investigated. The result of chromosome analysis indicated that 90% ploidy of one cell lines maintained normal when cultured up to passage 10. We compared the effects of cell cycle synchronization treatment of fetal fibroblast cells into G0/G1 by serum starvation or contact inhibition. There were no significant difference in percentage of G0/G1 between two treatments, no significant difference in percentage of G0/G1 by serum starvation for 2d and 4d, as the same when by contact inhibition.Serum-starved fetal fibroblast cells or cycling growing subconfluenced cells, round and smooth vs rough surface cells, different types of somatic cells and fibroblast cells from different individuals were used as donor in porcine somatic cloning. The results indicated that serum starvation could not improve the development of cloned embryos. Round and smooth cells led to a high fusion rate. Different cell types of donor and individuals affected the development of cloned embryos.A porcine fetal fibroblast (PFF) cell lines were transfected with pEGFP-N1 plasmid DNA containing both green fluorescence protein (GFP) and neomycin resistance genes under the control of CMV promoter by lipofectin-mediated way. Transgenic cells were selected as donor cells to construct cloned embryos, with blastocyst rate of 5.9-12.5%. Compared to non-transfected cells, there was not significantly different in the blastocyst rate.We evaluated a novel maturation medium, porcine zygote medium (PZM-3). Although it had a low maturation rate, there were not significantly different compared with North Carolina State University-23 (NCSU-23), NCSU-23 is a more successful maturation medium. There was not significantly different in NCSU-23, PZM-3 under 7%O2 concentration compared with 20%O2 concentration. Low oxygen concentration during IVC improved blastocyst rate and total cell number compared with high oxygen concentration regardless of culture media.The antral follicles of 3-4 mm diameter were aspirated. Grade A cumulus-oocytes complexes (COCs) with uniform cytoplasm and at least 3 layers of compact cumulus cells were selected and cultured. Oocytes with intact cytoplasm membrane, uniform cytoplasm and clear perivitelline space were chosen to be activated by electrically. Parthenogenetically activated oocytes, 2-embryos/μLmedium were cultured in PZM-3 with 3mg/ml BSA and 7%O2: 5%CO2: 88%N2. Blastocyst rate and total cell number were improved significantly. Optimization of nuclear transfer technique was as follows: At the 42 h of oocytes maturation, Oocytes were enucleated by aspirating the first polar body along with adjacent cytoplasm. Reconstructed couples were fused and activated in 0.1 mM Ca2± fusion medium.This is the first time that in vitro maturation oocytes in 7%O2 were made as recipient cytoplasm of nuclear transfer. Compared with 20%O2, blastocyst rate and total cell number of cloned embryos were improved significantly. A systematic study had been investigated on the effects of oxygen concentration (7%O2 vs 20%O2) and NCSU-23 , PZM-3 and G3 on the development of cloned embryos. These results indicated that blastocyst rate and total cell number were improved at low oxygen concentration. Among them, NCSU-23 in 7 %O2 greatly improved the blastocyst rate and cell num...