Construction Of Dppa5a Knockout Mice Using The CRISPR/Cas9 Gene Editing Technology And Analysis Of Phenotype

The developmental pluripotency maintenance factor 5A?Dppa5a?is a marker gene for pluripotent cells.It is specifically expressed in preimplantation embryos,germline cells,and embryonic stem cells?ES?,and it may mediate RNA transcription,regulate oogenesis,and embryonic development.CRISPR/Cas9 is an emerging gene editing technology that can cleave genes efficiently.In this study,Dppa5a^-/-mouse model was constructed by microinjection using CRISPR/Cas9 technology to analyze the phenotype,thereby studying the role of this gene in mouse growth and development.1.Expression Analysis of Dppa5a in Oocytes and Preimplantation Embryos of Wild Type MiceTo analyze the expression of Dppa5a gene in oocytes and preimplantation embryos by Quantitative Real-time PCR.The results showed that in germinal vesicle?GV?-stage oocytes,the expression of Dppa5a gene was significantly lower in oocytes of SN configuration than that in NSN.And the difference was extremely significant?p=0.0075?.In preimplantation embryos,large amounts of RNA accumulated in the metaphase II?MII?-stage began to drop dramatically after fertilization,reaching a minimum of only 9.0%in the 2-cell stage,and then slowly accumulating,and peaked in morulae,and it was 3.83 times that of the MII-stage.2.Construction of Dppa5a Knockout Mouse ModelTwo SgRNAs were designed on the second exon functional domain of the Dppa5a gene,and SgRNA was obtained by in vitro transcription.SgRNA was microinjected into pronuclear embryos,then cultured in vitro to 2-cell stage and embryo transfer was underway.After microinjection of 158 fertilized eggs,135 embryos were transplanted and were obtained 25 pups.Among them,six mice were found to have mutations at position 2 and the mutation rate was 24.0%.The F0 knockout mouse four deaths shortly after birth,so that we chose the 11th and15th mouse to breed.We used F0 generations to mate with WT to obtain Dppa5a^+/-heterozygous mouse during reproduction,and they were mated to generate Dppa5a^-/-homozygous mouse.These Dppa5a^-/-homozygous mouse was used to breeding homozygous mouse.In this experiment,a total of 188 Dppa5a^-/-knockout mouse come from No.11 were obtained for subsequent phenotypic analysis;it has 15 of the Dppa5a^-/-knockout mouse come from No.15 were used for cryopreservation of embryos,70 embryo of 2 cell and 10embryo of blastocyst were frozen.3.Phenotype analysis of Dppa5a^-/-miceBy analyzing the viability,growth ability and reproductive ability of Dppa5a^-/-mice,it was found that the absence of Dppa5a gene neither did it affect the viability,growth ability and reproductive ability of mice,nor did it affect the female-male ratio of the born mice.However,analysis of the small intestine revealed that the length of the small intestine was significantly shorter in the knockout mice,the intestinal mucosa had a certain range of necrosis and dissolution,and there was an intrinsic layer edema in the intestinal villi that did not dissolve the intestinal mucosa,and the muscle cells had local necrosis.Furthermore,the expression analysis of GLP-2R,Lgr5 and Pinin genes which related to the growth and development of intestinal mucosa showed that the expression of these three genes was down-regulated.It was suggested that Dppa5a gene might affect the development of small intestine by inhibiting the expression of GLP-2R,Lgr5 and Pinin.Thus,the results provide a theoretical basis for studying the function of Dppa5a gene.The Dppa5a knockout mouse model we constructed also provides an important biological model for Dppa5a gene and related research.