Effects Of TRIM28 On Imprinted Gene DNA Methylation In The Early Sheep SCNT Embryo

Objective:Due to incomplete genomic reprogramming and aberrant methylation, the somatic cell nuclear transfer(SCNT) embryos have many problems during development. Such as abortion, stillbirth and Large offspring syndrome(LOS)et al, Which limited the application of SCNT technology in animal husbandry. During early embryonic development, Trim28 plays an important role in DNA methylation maintenance of imprinted gene. The lack of maternal Trim28 is lethal to the normal fertilized embryos, and during the development process the embryos have the same syndromes like the abnormal fetuses of SCNT. Therefore,it is inferred that trim28 also play an important role in SCNT embryos’ genomic methylation.In order to reveal Trim28 gene’s function in development process of cloned embryo. This study will detect trim28 expression in matured oocytes after nuclear remove and imprinted genes methylation change in cloned embryos. And the purpose of this study is to provide an access to improve success rate of animal SCNT.Methods: Experiment one: China Merino sheep ovarian tissue was used to extracting RNA,and then reverse transcription to c DNA. The specific primers were designed to amplify the encoding region of TRIM28 gene and the cloned sequence was analysed by bioinformatics software. Our study used RT-PCR to detect the expression of TRIM28 in different tissues of sheep, and quantification of its interacted gene involved in DNA methylation regulation was tested. Experiment two: We have constructed TRIM28 eukaryotic expression vector and si RNA. The TRIM28 gene m RNA and protein were measured by RT-q PCR and Western Blot in sheep fibroblast cell with electroporation method, and the expression of DNA methylation related gene and imprinting gene was quantificated. Experiment three: Normal somatic cells(N-SCNT) and the TRIM28 overexpression cells(H-SCNT) were used to SCNT, then collected cloned embryos in different developmental stages. TRIM28 gene m RNA expression was assembled in N-SCNT and H-SCNT. The 2-cell and blastocysts were collected for extracting genomics and cloning of imprinted gene H19 and IGF2 R DMR by BSP bisulfite treatment.Results: we cloned the complete CDS sequence of TRIM28 gene in sheep, the length of the nucleotide sequence of 2441 bp. Both the c DNAs encode proteins of 824 acid residues,showing high sequence identity with human, cow, pig, mouse et.al. TRIM28 is expressed in sheep’s all tested tissues, such as heart, liver, spleen, lung, kidney, muscle, ovarian. Tissue expression profiles showed that TRIM28 m RNA is abundantly expressed in China Merino sheep lung, spleen and ovary, and the level is consistent with ZFP57 in all detected tissues.We have successfully constructed the sheep TRIM28 eukaryotic expression vector p EGFP-C1-TRIM28, and efficiently expressed in sheep fibroblast cells with electroporation method. We have also selected a si RNA, which can effectively inhibit the expression of TRIM28 in sheep fibroblast cells.Changing the TRIM28 expression level lead to DNA methylation related gene DNMT1、DNMT3B、ZFP57、SETDB1 down regulated,but there was no significant effect on the DNMT3 A expression. The expression of imprinted gene IGF2、IGF2R、PEG1.2、PEG3、MEG8 is consistent with expression of TRIM28, while H19 and DLK1 were down regulated whether the TRIM28 level up or down.Conclusion:The Sheep TRIM28 gene complete encoding region sequence have high similarity with other species.Our results suggest that TRIM28 plays an important role not only in the methylation of imprinted genes in early embryonic development, but also in highly differentiated fibroblast cells. TRIM28 overexpression somatic cell can effectively improve the TRIM28 level in early SCNT embryo, imprint gene H19 and IGF2 R methylation level higher than the normal SCNT group. These foudings seem to indicate that TRIM28 is an important factor for the methylation abnormality of sheep cloned embryos.