Regulation Of DNA Methylation And Imprinting Gene Expression In Early Pre-Implantation Cloned Mice

In order to disti(?)uish whether the U2afbp-rs gene which is expressed in clonedmice embryos is fro(?)paternal or from maternal, I should find out a polymorphismsite in U2afbp-rs g(?)e between different mouse lines. In order to find out thepolymorphism of U2(?)bp-rs gene between different mouse lines including Kunming,B6, Balb/c, 129S1, (?)H, DBA and A/J, single strand conformation polymorphism(SSCP) was used (?)detect the possible polymorphism sites; Embryos that wereproduced by parthe(?) genesis were cultured to morula, and then each cell of morulaswas transferred into (?)cleate MII occytes, as called parthenogenetic morula nucleartransplantation emb(?) os here; NIH3T3 cell nucleus were also transferred intoenucleate MII ooc(?)s. In order to prove up the relationship between nuclearreprogram and DN(?)ethylation during the process of nuclear transplantation, thetwo cloned pre-imp(?)tation embryos together with in vitro fertilization (IVF) andparthenogenetic em(?)os were subjected to immunocytochemistry with antibody toDNA methylation((?)MeC). In order to research the regulation of oocyte cytoplasmto the relative expr(?)on level of imprint genes, we detected the relative expressionlevel of U2afbp-rs (?)e which is maternally imprint and non-imprint eIF-4C gene inpre-implantation (?), parthenogenetic and parthenogenetic morula nucleartransplantation emb (?)s by real-time PCR. The results are showed as follows:1. Although th(?) are several polymorphism sites between C57BL/6J and PWKmouse, which is a (?)ilable abroad, there is no polymorphism of these sites indomestic mouse li(?) mentioned above.2. Imprinting (?)e U2afbp-rs and non-imprinting gene eIF-4C are expressednormally in IVF (?)se embryos, but in parthenogenesis, both of them expressabnormally, which (?)y be caused by the remodeling of chromatin conformation andDNA methylation i(?) arthenogenesis.3. Both the re(?)ive expression level of U2afbp-rs gene and eIF-4C gene inparthenogenetic m(?)la nuclear transplantation embryos are lower then control parthenogenetic embryos, but the expression pattern of the two genes were similar tothe control parthenogenesis group. The results suggest that the imprinting geneexpression of donor nuclear were regulated by oocyte cytoplasm, but the regulationis not completed.4. Neither the genomic DNA methylationlevel of NIH3T3 cloned embryos norparthenogenetic morula nuclear transplantation embryos was changed markedlybetween pre- and after- nuclear transplantation. The results suggest that the activedemethylation of the donor nucleus genomic DNA is not completely.