DNA Methylation Profiles Of Imprinted And Putative Imprinted Genes On Somatic Cell Nuclear Transfer Sheep

Somatic cell nuclear transfer (SCNT) has been successfully applied to many mammalian species. In spite of these remarkable achievements, this valuable technique has raised many questions such as increased abortion rate, perinatal death, low pregnancy rates and increased fetal and placental abnormalities, named as large offspring syndrome(LOS). Till recently, abnormal epigenetic modifications, such as DNA methylation, histone modifications and chromatin reconstructions have been reported in several cloned species. In the present study, in order to show the reprogramming degree of cloned lambs, we investigated DNA methylation patterns in CpG islands and DMR of eight imprinted and putative imprinted genes in five tissues of cloned lambs.BSP and MSP were used as the methods of DNA methylation analysis in this study. Five tissues of two cloned lambs died after birth and two natural produced lambs were investigated, including lung, heart, kidney, liver and muscle. Moreover, several fragments of putative imprinted genes were purified and submitted to Gene Bank. mRNA expression profiles of Igf2r,Dlk1,Xist and Peg10 in five tissues of cloned lamb D2 were also examined in this study. The results were as follows:(1)A 1163bp fragment of ovine Peg10 was purified(GI:269838596),a 848bp partial cDNA of ovine Peg10(GI:285014443),a 764bp 5'region of ovine Xist, a 483bp partial cDNA of ovine Xis(tGI:285014445) and a 307bp partial sequence of ovine Peg3.(2)A 205bp fragment in CpG island of ovine Peg10 was differential methylated.(3)Eleven consecutive CpG sites in CpG island of ovine Peg3 were all methylated in tissues of ovine lung and kidney.(4)Nineteen consecutive CpG sites in CpG island of ovine Cdkn1c were all nonmethylated in tissues of ovine lung and kidney.(5)DNA methylation pattern of seventeen consecutive CpG sites in CpG island of ovine Dlk1 was mosaic in all tissues. It was not a differential methylated region.(6)DNA methylation pattern of six consecutive CpG sites in CpG island of ovine H19 was mosaic in all tissues. It was not a differential methylated region.(7)The DNA methylation of putative imprinted genes of cloned lamb D1,D2 were nearly same as controls,and there were not evident differences between cloned lambs and controls except that the DNA methylation of Dlk1 and H19 in few tissues of cloned lamb D2 showed higher Methylation. (8)The MSP results of Xist showed that the analyzed region of ovine Xist gene is a putative differential methylated region and there were not evident differences of DNA methylation pattern between cloned sheep and controls.(9)The RT-PCR results of ovine Peg10 and Dlk1 exhibited that their expression were tissue specific.(10)The mRNA expression of Peg10 and Dlk1 in newborn lamb were weak than other examined genes.(11)The mRNA expression of Xist,Peg10,Dlk1 and Igf2r in tissues of cloned lamb D2 were nearly same as controls.In a word,there were not evident differences of DNA methylation and mRNA expression profiles between cloned lambs and controls. And somatic cell nuclear transfer sheep experienced relatively completed DNA methylation reprogramming.