Preliminary Study On The Function Of P400 In Mouse Pre-implantation Embryo And Embryo Fibroblast

p400 encoded by Ep400 was identified as a novel adenovirus E1 A binding complex,which is a SWR1-class DNA-dependent chromatin remodeling ATPase,containing a bipartite SWI2/SNF2-type ATPase domain,a SANT domain,an HAS domain and a glutamine-rich domain.p400 plays an important role in DNA damage,histone replacement,cell cycle and post-implantation embryo development.However,little is known about the function of p400 during mouse preimplantation embryo development.Therefore,the present study focuses on exploring the function of p400 in the development of preimplantation embryos in mice.1.The dynamic changes of p400 in pre-implantation embryos at various stages in mice.Pre-implantation embryos in five stages of mouse zygote(10h),2-cell(24h),4-cell(48h),morula(72h),and blastocyst(96h)and mouse germinal vesicle(GV)eggs were collected and the expression of p400 protein was dectected by immunofluorescence Staining technique.The results showed that p400 was expressed in all stages of pre-implantation embryos in mice,and was mainly expressed in the nucleus.With the development of pre-implantation embryos in mice,the expression level of p400 gradually increased from the GV period before fertilization to the 2-cell period after fertilization,the expression level decreased during the 4-cell period,and it increased again during the morula embryo to blastocyst period and reached the highest expression level in the blastocyst stage.Total mRNA of p400 in various stages of preimplantation embryo development in mice was detected by real-time fluorescence quantitative PCR.No significant change in p400 mRNA levels in zygote stage compared to it in GV stage.p400 expression levels increased in 2-cell stage,decreased to levels in GV stage again in 4-cell period.And continued to decline during the blastocyst to morula stage.The p400 mRNA expression level decreased to a minimum in the blastocyst stage.2.Construction of Ep400 gene knockout cell lines.In order to further study the effect of p400 on mouse preimplantation embryo development,CRISPR-Cas9 technology was used to knock out the Ep400 gene in mouse embryonic fibroblasts to explore its effect on cell growth and development.The knockout vector was constructed,and the Ep400 gene was knocked out by the method of single site and double site.Double-site knockout used sgRNA on bouth side of the selected exon of Ep400 effectively knock out the Ep400 gene by forming fragmented deletions.Finally,after the Ep400 gene was successfully knocked out,the mouse embryonic fibroblast monoclonal cell line with the Ep400 gene knocked out was screened.Using Ep400 gene knockout cell lines to explore the effect of p400 on the growth of mouse embryonic fibroblasts,and further lay the foundation for exploring the effect of p400 on the development of mouse pre-implantation embryos.3.The effect of knocking down Ep400 gene on the proliferation of mouse embryonic fibroblasts.The shRNA was used to knock down the Ep400 gene,and its effect on the proliferation ability of NIH 3T3 cells was detected by the CCK-8 experiment.The results showed that after knocking down the Ep400 gene,mouse embryonic fibroblasts showed a decrease in proliferation capacity after 48 h.