The Effect Of Small Molecule Treatment On The Development Of Golden Snub-nosed Monkey-goat Inter-species Nuclear Transfer Embryos In Vitro

Golden snub-nosed monkey is listed as national level endangered wild animal.Faced with the dramatic decline in the number of surviving monkeys,how to optimize their growth,development and reproductive breeding problems has caused widespread attention.Due to the limited number of species in the same type of nuclear transfer,inter-species nuclear transplantation can successfully avoid these problems.Inter-species nuclear transplantation is affected by problems such as nuclear transfer process,receptor cell and donor cell selection,especially donor cell reprogramming capability.Therefore,the treatment of donor cells play a role in the success of inter-species nuclear transplantation.With the development of somatic cells pluripotency,It has been widely applied to improve the reprogramming efficiency by using small molecular compounds to induced pluripotency epigenetic modification of donor cells.In this experiment,we have golden snub-nosed monkey donor cells treated with small molecular compounds of epigenetic modification,with goat oocytes as recipient,to explore the influence of different treatments on in vitro development of inter-species nuclear transplantation embryos.The experimented investigation and the results are as follow:The research materials were the golden snub-nosed monkey ear skin tissue,and fibroblast cells were isolated by tissue block method.The cells were treated with appropriate concentration of small molecular compounds and the cell morphology,proliferation ability and the expression level of the pluripotent gene was detected:We successfully isolated the monkey ear skin fibroblast cells,after several passages cells can still maintain high activity;PCR showed the expression of Sall4 and Gata6,which is expression of the extracellular endoderm;Q-PCR showed the expression of Sall4 and Gata6 increased.Gata6 expression suddenly accelerated on day 2?day 4 and day12(P<0.05),and in the 6th and 14th days were extremely significantly increased(P<0.01).Sall4 increased obviously after treatment,expression increased significantly on days 2.day 4 and day 10(P<0.05),and in the 6th?8th?12th and 14th days were extremely significantly increased(P<0.01).The golden snub-nosed monkey fibroblast cells and goat oocytes fused to form recombinant embryos.The development of embryos and rate was observed,the expression of early growth related genes was detected by Q-PCR and immunofluorescence staining:There was no significant difference between the treated group and the control group in the whole development process(P>0.05);Expression level of Oct4 and Prrxl significantly increased(P<0.05),and the expression level of Sox2 and Telemeras were extremely significantly increased(P<0.01).On the contrary,the expression level of Nanog was decreased(P>0.05);OCT-4?SOX-2 and NANOG protein expression are detected by immunofluorescence staining,but there were no significant differences between the two groups(P>0.05).This study discovered that the treatment of small molecule compounds can significantly improve the expression of pluripotent marker genes,which laid the foundation for further research.