Expression And Methylation Status Of Imprinted Genes In Cloned Pig

Pig organs may become the sources of organs for mankind in the future. Although studies of somatic cell nuclear transfer and transgene on pig have made some progress, the efficiency of nuclear transfer is still very low (1% ~ 2%). Imprinted genes play an important role on the growth and development of mammals, in natural breeding mammals, imprinting is established during gametes formation by changes of the methylation status, however, cloned mammals pass this stage, and directly development by injection of a somatic cell into an enucleated egg, which may cause abnormal expression of imprinted genes. This study was focus on the expression and methylation status in cloned pig and their embryos, and it provides a theoretical basis on improving the efficiency of porcine somatic cell nuclear transfer. The major findings are as follows:(1) We analyzed expression levels of four imprinted genes (IGF2,H19,PEG3,GRB10) in live cloned piglets,dead cloned piglets,natural breeding piglets and their corresponding placentas by Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we found that the expression of all four imprinted genes (IGF2, H19, PEG3, and GRB10) was significantly reduced in placentas of dead clones compared with placentas of live cloned piglets and controls ( p<0.05). In contrast, both live and dead cloned piglets exhibited steady-state mRNA levels for these genes within the control range (p>0.05). Transcript levels for these genes in live clones rarely differed from those of controls in both piglets and placentas.(2) Examination of the methylation status of DMR2 of IGF2 and CTCF3 of H19 genes revealed that both genes exhibited significant high methylation levels in placentas of dead clones compared with placentas of liveclones and controls. In contrast, both genes showed a normal differential methylation pattern in live cloned pigletsand their placentas compared with controls. Importantly, dead cloned piglets also showed a normal pattern. Our results suggest that abnormal expression of imprinted genes in placenta may be caused by abnormal methylation patterns in differentiallymethylated regions (DMRs) of imprinted genes.(3) Examination of the methylation status of DMR2 of IGF2 and CTCF3 of H19 genes revealed that both genes exhibited demethylation in nuclear transfer embryos compared with in vitro fertilized embryos. Furthermore, demethylation of both genes happens in 6 to 15h of one cell stage and two cell stages, which may be brought by procedure of SCNT. (4) We separated inner cell mass and trophoblast of in vitro fertilized and NT embryos by immunosurgery; we confirmed our results by analysis of marker gene of inner cell mass and trophoblast respectively.(5) Examination of the methylation status of DMR2 of IGF2 and CTCF3 of H19 genes revealed that both inner cell mass and trophoblast exhibited abnormal methylation levels in nuclear transfer embryos compared with in vitro fertilized embryos. Howerver, methylation levels of inner cell mass is more normal than trophoblast, indicating that cells with errors of methylation imprints preferrly participate in the trophoblast rather than inner cell mass, which maybe the reason of abnormal methylation imprints in placenta.(6) We examined DNA replication time of one cell stage of NT embryos by BrdU labeling method, and found that DNA replication occurred during 6 to 15h in one cell stage NT embryos. The results suggest that demethylation of methylation imprints in NT embryos is caused by failure of maintence methylation during DNA replication.