The Role Of Orc6 Gene In Mouse Preimplantation Embryo Development

The replication recognition complex 6?Orc6?is a protein-related to the initiation ofDNA replication and is the only subunit of the replication recognition complex?ORC?that is not related to AAA+ATPase.At present,the Orc6 plays a role in the process of DNA replication,chromatin distribution,spindle assembly,etc.,but its role in embryo development is unclear.By the Cre-LoxP system,we found that homozygous embryos with Orc6 gene knockout were dead.In this experiment,a model of Orc6 embryo knockdown was established by cytoplasmic injection through a microinjection system to explore the role of Orc6 in the mouse early embryo development before implantation.1.Orc6^-/-embryo phenotype analysisBy taking the offspring of E7.5 and E3.5 for phenotypic analysis,we found that the genotype ratio of E7.5 was imbalanced,but the genotype ratio of E3.5 embryos confirmed to Mendel's law.It is speculated that the death time of Orc6^-/-embryos is at E7.5-E3.5,followed by outgrowth culture of E3.5 embryos,homozygous embryos did not break through the zona pellucida.2.Orc6 mRNA expression in early embryos before implantationUsing quantitative PCR to analyze the expression of Orc6 in oocytes and early embryos,it was found that the expression of Orc6 was irregular at all stages of embryo development.From the oocyte to the fertilized egg stage,the expression of Orc6 mRNA increases significantly,the 2-cell stage decreases,the 4-cell,and 8-cell stages increase,the expression of the mulberry embryo stage gradually decreases,and the lowest level at the blastocyst stage.3.Effect of Orc6 gene silencing on early embryo development before implantationOrc6 siRNA was injected into the cytoplasm to establish an embryo knockdown model.Through immunofluorescence and quantitative PCR,we learned about the effects of Orc6 on the fate of the first and second cells in mouse embryonic development,and on apoptosis and spindle assembly.The experimental results suggest that Orc6 affects the fate distribution of PrE cells by affecting the expression of Sox17,a regulator that determines the fate of PrE cells.Besides,silencing the Orc6 gene can cause irreparable DNA damage in the embryo.But the Orc6 gene silencing did not affect spindle assembly.In summary,Orc6 gene silencing affects the fate distribution of PrE cells and causes irreparable DNA damage,but does not affect the correct assembly of the spindle.Through experiments,we have a further understanding of the function of the Orc6 gene,which fills in a new idea for the study of the fate decision of the second cell during embryonic development.