Effect Of Pold2 Gene On Preimplantation Embryo Development In Mouse

Pold2 gene,a subunit of DNA polymerase?complex,encodes a protein involved in DNA replication and repair.DNA replication is very important for the normal life activities of cells,which is not only a simple DNA duplication,but also to maintain the integrity of the genome in the process of damage repair.Studies have shown that pold2gene can affect cell proliferation and is highly expressed in a variety of cancer cells.Therefore,most of the studies on pold2 gene are focused on these aspects,and little is known about its function in mammalian embryo development.Therefore,In this study,Cre-lox P system and RNAi technology were used to study the role of pold2 gene in the development of mouse preimplantation embryos.The results showed that the homozygous mutant embryos with pold2 gene knockout died,and the death time was between E 3.5 and E 7.5.Then,established the model of pold2 gene knockdown embryos,and used knockdown embryos instead of knockout embryos to study the function of pold2 gene in the development of mouse preimplantation embryos.1)Phenotypic analysis of pold2^-/-embryosBy genotyping the mouse embryos obtained at E 7.5,it was found that there was no homozygous mutant(Mut)embryos,which indicated that the mutant was not successfully implanted into the uterus.However,the Mut embryos collected at E 3.5were not abnormal in morphology compared with wild-type(WT)and heterozygous(Het)embryos.It is speculated that the death time of pold2^-/-embryo is between E 3.5and E 7.5.Then,the embryos at E 3.5 were cultured in outgrowth culture.The results showed that the mutant embryos could not hatch.These results suggest that pold2^-/-embryos can form normal blastocysts,but cannot be implanted into the uterus for further development.2)Expression and localization of pold2 in mouse oocytes and preimplantation embryosRT-q PCR was used to detect the m RNA expression of pold2 in mouse oocytes and preimplantation embryos,and detected its protein expression level and location by embryo immunofluorescence.The results showed that the m RNA expression of pold2gene was found from oocytes to blastocysts,but there was no rule to follow,and its protein expression corresponded to m RNA expression.In addition,POLD2 protein was localized in the nucleus except in the cytoplasm of MII phase.3)Effect of pold2 gene knockdown on preimplantation embryo developmentPold2 knockdown embryo model was successfully established by injecting pold2si RNA into the cytoplasm of mouse zygote.The effects of knockdown on the embryo development before implantation were analyzed by outgrowth and immunofluorescence.The results showed that knockdown of pold2 gene did not affect the formation of blastocysts or the first lineage differentiation of blastocysts.However,Outgrowth experiment failed.During the second lineage differentiation of blastocysts,the number of Sox17 positive cells decreased significantly,even about half of the embryos could not detect the fluorescence signal of Sox17 directly,and the proportion of Sox17 positive cells in the total cells also decreased significantly.These results indicate that the knockdown of pold2 gene affects the second lineage differentiation of embryo,and then leads to implantation failure.In conclusion,this experiment first found that pold2 gene is crucial to the development of preimplantation embryos in mice.Knockdown of pold2 gene does not affect the formation of blastocysts,but it will lead to the failure of blastocysts to break through the zona pellucida and then normal implantation into the uterus.It is also proved that the gene can affect the second lineage differentiation of embryo.This study provides some new ideas for the study of mouse embryo implantation and lineage differentiation,and lays a theoretical foundation.