Comparison Of Genomic-wide Methylation Atterns Preimplantation Embryos And Al Analysis Of Dnmts Promoter Of Mouse

In order to investigate the influences of in vitro fertilization, operation and culture environment on mouse IVF preimplantation embryos, the DNA methylation patterns of in vivo fertilization and IVF mouse preimplantation embryos were investigated by immunofluorescence staining with an antibody against5-methylcytosine (5-MeC). Using In vivo fertilized preimplantation embryos as the control, we investigated genomic-wide DNA methylation patterns of in vivo fertilization and IVF mouse preimplantation embryos. The result showed that IVF mouse preimplantation embryos had different genomic-wide DNA methylation pattern compared to in vivo fertilization mouse preimplantation embryos. The methylation level was higher in pronuclear stage, reduced in2-,4-,8-cell stage and slightly higher in morula and blastocyst stage. The DNA methylation levels of IVF embryos at different developmental stages were lower than those of in vivo fertilization embryos. In vitro fertilization, manipulation and culture environment might affect the DNA methylation pattern and result in abnormal methylation pattern of in vitro fertilization embryos. The promoter as the gene expression switch is an important cis-acting elements.it can be ragulate by trans-factors to control turning on and of of gene expresion. In the this study, we cloned the Dnmtl,3a,3b gene promoter of mouse and constructed Dnmtl,3a and3b promoter5’deletion fragments(-pGL3)we called them Dnmt1-1、2、3、4, Dnmt3a-1、2、3、4, Dnmt3b-1、2、3、4. At the same time, we constructed the transformation of ox2、Oct4、 Nanog-pcDNA3.1、Klf4-pcDNA3.1、c-Myc-pcDNA3.1. We co-transfect every promoter and transcription factors into HKE293cell and detect the relative activity of the luciferase by dual luciferase rreporter system. By this method, we have investigated the influence of transcription factors to methyltransferase promoter activity. The results show that:five transcriptions inhibit on activity of Dnmt1-1、4more strongly than on activity of Dnmt1-2、3. Five transcriptions factors inhibit on activity of all of4Dnmt3a promoter5’deletion fragments but every transcriptions factors have different degree of inhibition on every Dnmt3a promoter5’deletion fragments. Nanog have more strong inhibition on activity of Dnmt3a-1than on others and inhibition on activity of Dnmt3a-2、3、4have no significant difference. transcription factors Oct4、c-Myc mainly inhibit the region between Dnmt3a-1promoter fragment and Dnmt3a-2promoter fragment. Inhibition Sox2on4Dnmt3a promoter fragmen is not obviously different. Five transcription factors can significantly promote the activity of Dnmt3b-2,3promoter fragment, whose activity is more than7times higher than control. But5transcription factor is almost no effect on the activity of Dnmt3b-1,4promoter fragment, their luciferase activity are equal to the control group.