The Research Of Production Of Porcine Transgenic Embroys By Intracytoplasmic Sperm Injection

The study of animal transgenic technology which could modified livestock genome and had significant effect to improve livestock production was begun from1970s.The types of transgenic technology had prenuclear microinjection method, the sperm mediated method, retrovirus vector infection method and somatic nuclear transfer method, etc. Successful offspring production after a new technique which was intracytoplasmic sperm injection(ICSI) combined with Sperm mediated Gene Transfer(SMGT) had been reported in many laboratory animals, and ICSI-SMGT proceduce for production of transgenic animals was feasible. As a vector, Bacteria Artificial Chromosome(BAC) has played an important role on carrying the genome sequences, including upstream and downstream regulation sequence, transcriptase and transcription factors, etc. And the expression of genome will be able to observe the gene expression of the inherent mechanism. Thus, the ICSI-SMGT technology combined with BAC transgenic technology(ICSI-SMGT-BAC) was a new method for production of large DNA fragmentations of porcine transgenic embryos, it had broad application prospects. For preparing porcine transgenic embryos successfully, this study will establish an effective system which can conserve the immature porcine oocytes in vitro more than6hours and achieve the goal of production of porcine transgenic embryos by ICSI-SMGT-BAC.First, establish the system of conserving the immature porcine oocytes in vitro. To selected the suitable medium and temperature for conserving the oocytes in vitro, we examined the effect of conserved immature porcine oocytes in vitro on maturation ability of oocytes and developmental capability of embryos after in vitro fertilization. First, we selected three medium which were TCM199(with HEPES), D-PBS and DMEM(low sugar) to conserve the oocytes6-8h, and the immature oocytes following in vitro maturation(IVM), in vitro fertilization(IVF) and in vitro culture(IVC). We recorded the data of Mature rate, Cleavage rate and Blastula rate, and then compared this three mediums. The results showed the control group mature rate was78.19%, the cleavage rate was55.87%and blastula rate55.87%; TCM199group mature rate was76.25%, the cleavage rate was55.00%and the blastula rate was12.12%; The PBS group mature rate was49.00%, the cleavage rate was22.00%and the blastula rate was2.27%; DMEM group mature rate was47.50%, the cleavage rate was20.50%and the blastula rate was2.43%. It showed there were not significantly different between the control group and TCM199group. And the TCM199group was significantly higher than D-PBS and DMEM groups. For more, we carried out the experiment of embryo transfer(the embryos were from TCM199group), and the pregnancy sow had given birth to11piglets which were in good health. So TCM199can be used as a medium for conserved oocytes in vitro. In order to determine the suitable temperature, we select4℃,25℃and37℃, the medium was TCM199. The Mature rate, Cleavage rate and Blastula rate were recorded, and the three temperature were compared. The result showed4℃group mature rate was19.00%, the cleavage rate was9.5%and no blastula;25℃group mature rate was33.00%, the cleavage rate was15.00%and no blastula;37℃group mature rate was76.50%, the cleavage rate was54.50%and the blastula rate was13.76%. From the result, we think TCM199,37℃was the suitable condition for conserved oocytes in vitro.Second, to achieve the goal of production of porcine transgenic embryos by ICSI-SMGT-BAC. It is necessary to produce porcine embryos by ICSI. In this study, first, the sperm tail removed by sonication(3w, work3s, interval3s, total60s). Second, ICSI was conducted using sperm head, and then ICSI oocytes were stimulated with a direct current pulse of170V for90μs. Last, the embryos were cultured, and the stage of embryo development were record. The results showed we can provide technical basis to produce large fragments of transgenic embryos.This study establish an effective system to conserve the immature porcine oocytes in vitro more than6hours and explore possible to produce porcine transgenic embryos by ICSI-SMGT-BAC.