The Study Of Igf2Gene Abnormal Imprinting Patterns During Early Development Of Cloned Embryos

At present, that has been cloned a variety of mammals successfully, such as bovine, goats, pigs, mice, rabbits and so on. But the SCNT technology efficiency is very low, only about4%. A large number of studies had showed, as the donor cell nuclear, the highly differentiated state somatic cell nuclear have high level methylation. The incomplete reprogramming of donor cell nuclei leading to aberrant expression or lack of expression of some developmentally important genes has been implicated as a primary reason for the low efficiency of SCNT. It will lead to gene expression abnormality, especially, the genomic imprinting abnormality.Genomic imprinting is some of the alleles in the process of expression, select the expression maternal or paternal gene. The CpGs methylated is an important way in genomic imprinting regulation. The imprinting gene Igf2is a typical maternal imprinting gene. It has four promoters, the promoter one is biallelic express, but the other promoters are paternal allele expression. It has3differentially methylated regions(DMR), and the DMR1region is the key, the DMR1methylation status directly affects the Igf2gene occur gene imprinting.In this study, we collected five developmental stages of the bovine SCNT and IVF early embryos, each stage40. The five developmental stages include Zygote phase, S phase,2-cell phase,8-cell phase and morula phase. The detection of the DMR1methylation status of the Igf2gene use Bisulfite Sequence PCR(BSP). Study the Igf2gene overall methylation status and DMR1methylation locus of methylation status in the early embryos. Comparison methylation difference of Igf2gene in two early embryos to research abnormal genomic imprinting, obtained following conclusions:1. The methylation levels higher in the SCNT than the IVF early embryos. The DMR1region of Igf2gene methylation is abnormal, and it will causing Igf2gene imprinting abnormal.2. Comparing the overall methylation status of SCNT and IVF early embryos during five different periods. During the S phase, the mean methylation levels of Igf2in SCNT and IVF early embryos were36.2±2.96%and22.16±4.36%, differences significant(P<0.05). During the8-cell phase, the mean methylation levels of Igf2in SCNT and IVF early embryos were33.47×10.09%and24.3×2.51%, differences significant(P<0.05). During S phase and8-cell phase, the methylation status of Igf2are abnormal, S phase and8-cell phase are Igf2gene abnormal imprinting.3. Comparing the CpGs methylation status of SCNT and IVF early embryos during five different periods. In the IVF early embryos, the CpGs methylation pattern is complex. In the SCNT early embryos, the CpGs methylation pattern uniformity is better. In the18CpGs, the4,6,8,9,10methylation locus is susceptibility locus. These methylation locus is key of Igf2gene abnormal imprinting.