| Miniature pigs have been as a model animal for a long time.Compar ed to the mice and other models, miniatur e swine model have mor e s imilar ities to human in many points, such as weight, organ s ize, str uction of phys iology and anatomy, and also mechanism of disease, all of those make the miniatur e sw ine model to have a great value in the biomedic al r esearch. Many genes in r odent models do not have the s imilar functions of phys iological and biochemic al with the human body. So compared to the rodent model, miniature swine model closer to the people can better ver ify gene function in large animal model by studying. But befor e CRIS PR system appears, the limit of gene knockout in miniature sw ine is that ther e is no suitable porc ine embryonic stem cell lines for gene editing. CRISP R appears greatly f acilitates the emer gence of porc ine gene knockout models.PDX-1 gene plays an important role in pancreas development. In ear ly embr yonic development,pancreas der ived mainly through the epithelial bud f us ion of dorsal and ventr al s ides to form pancreatic bud. The homozygous of PDX-1 knockout mic e lack of matur e pancreatic tissue, and als o showed abnormal state in some part of the duodenum region.Thes e mice can not survive for a long time after bir th, it will soon die of hyperglycemia. I t is unc lear about the f unction of PDX-1 gene in Bama miniatur e because the lack of research on gene knockout models.In this study, high-quality embryos wer e collected from super ovulated Bama minature pig and injected w ith Cas 9 mRNA/sgRNA to pr oduce PDX-1 gene knockout pig.Traditional pig embryos obtained through in vitr o mainly I VF, NT and parthenogenetic.While it is hard to obtain good embyo in vitro to support development due to the imperfections of the swine in vitro culture system. But the embryo obtained by in vivo superovulation could be better than these in vitro. It can provide a stable platfor m for produc ing the knockout pig model.In summary of our data, we found that:( 1) The best procedure of superovulation in Bama is as followed :injection 11 I U/kg cloprostenol at 9:00 am in the 15 th day of estr us and repeat af ter 8 hours, followed by 21 I U/kg at 9:00 am in the next day. After 72 h, 16.7 I U / kg hCG is injected and after 24 h checking for estrus. F inally wash the f allopian tube to t ake embryos after 24 hours, it can be used to get a lot of quality embryo for intracytoplasmic microinjection.(2) The quality and amount of embr yos obtained by superovulation in vitro are both better than other ways( P<0.05), and has a good development ability to produc e nor mal pigs, which w ill be useful for obtaining the gene knockout animals model by CRISPR/Cas9 system.(3) PDX-1 gene knockout can lead to a lack of pig pancr eas organ. |
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