【作者】 赵丽霞；

【导师】 周欢敏；

【作者基本信息】 内蒙古农业大学 ， 动物发育生物学， 2010， 博士

【摘要】 体细胞核移植技术在医疗、农业及畜牧业等诸多领域有重要的应用价值。尽管利用该技术已经成功获得了多种克隆动物,但随之而来的克隆胚胎及个体成活率低、不同种属普遍存在的LOS(large offspring syndrome)等异常制约了体细胞核移植技术的广泛应用。目前,有许多学者认为不完全表观重编程是克隆效率低下的原因。本研究通过检测新生死亡克隆绵羊印记相关基因的DNA甲基化水平,探究克隆绵羊DNA甲基化的重编程程度。本研究主要采用重亚硫酸盐修饰测序法(BSP)和甲基化特异性PCR(MSP)法检测了2-3日龄死亡克隆蒙古绵羊肾脏、肺脏、肝脏、心脏和肌肉组织中8个印记相关基因的DNA甲基化水平与正常对照的异同,其中克隆个体羔羊2只,正常对照2只。此外,本研究进一步比较分析了Igf2r、Dlk1、Xist和Peg10在克隆绵羊D2和正常对照五种组织中的mRNA表达情况。在进行上述分析之前,本研究首先克隆了部分基因片段。通过实验得到如下结果:(1)本研究成功克隆到了绵羊Peg10 1163bp的拟DMR序列(GI:269838596),848bp绵羊Peg10部分cDNA序列(GI:285014443),764bp的绵羊Xist第一外显子5’端序列及483bp的cDNA序列(GI:285014445),307bp的绵羊Peg3第一外显子5’端序列。(2)绵羊Peg10 5’端CpG岛内205bp范围内区域为差异甲基化区域。(3)绵羊Peg3外显子5’端CpG岛内232bp范围内11个CpG二核苷酸对上的胞嘧啶在2-3日龄绵羊肾脏和肺脏中基本完全甲基化。(4)绵羊Cdkn1c外显子5’端CpG岛内188bp范围内19个CpG二核苷酸对上的胞嘧啶在2-3日龄绵羊肾脏和肺脏中基本非甲基化。(5)绵羊Dlk1转录起始位点上游CpG岛内188bp范围内17个CpG二核苷酸对上的胞嘧啶甲基化模式为马赛克式,该区域并非差异甲基化区域。(6)绵羊H19启动子区CpG岛内CTCF结合位点III附近121bp范围内6个CpG二核苷酸对上的胞嘧啶甲基化模式为马赛克式,该区域并非差异甲基化区域。(7)克隆绵羊D1、D2各组织各印记相关基因的DNA甲基化模式与正常对照基本相同,无明显差异,仅D2个别组织Dlk1甲基化水平略高于正常对照,D2 H19肝组织的甲基化水平高于对照。(8)克隆绵羊Xist MSP甲基化结果显示绵羊Xist分析区域可能为差异甲基化区域,而且克隆绵羊的甲基化模式与正常对照无显著差异。(9)半定量RT-PCR结果显示绵羊Xist和Igf2r基因表达基本无组织差异性,而Peg10的mRNA表达表现组织特异性,肾脏中表达水平强于其他组织。(10)Peg10和Dlk1在新生个体组织中的mRNA表达水平比其他检测基因弱。(11)克隆绵羊D2肾脏、肺脏、肝脏、心脏和肌肉组织中Xist、Peg10、Dlk1和Igf2r基因mRNA的表达水平与正常对照相比,差异不显著。综上,本研究认为2只体细胞克隆绵羊的DNA甲基化水平与自然生产绵羊无显著差别,即体细胞核移植克隆绵羊经历了比较完全的DNA甲基化重编程。同时,克隆个体上述基因的mRNA表达水平也未表现出明显异常,暗示体细胞核移植克隆绵羊经历了比较彻底地重编程。更多还原

【Abstract】 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.更多还原