克隆山羊成纤维细胞IGF2-H19基因座甲基化分析

发布时间：2018-03-20 23:03

本文选题：体细胞核移植　切入点：再克隆　出处：《畜牧兽医学报》2017年12期 　论文类型：期刊论文

【摘要】：旨在研究山羊体细胞核移植对克隆后代成纤维细胞IGF2-H19基因座甲基化的影响。以奶山羊耳成纤维细胞(GFC,对照组)和克隆山羊耳成纤维细胞(CFC,试验组)为试验材料,培养至第5代时,采用细胞计数法绘制细胞生长曲线,流式细胞仪检测细胞凋亡情况,实时荧光定量PCR分析基因的表达差异,亚硫酸氢盐测序(BSP)分析差异甲基化区域的甲基化水平。结果显示,GFC和CFC组细胞的生长曲线均呈典型"S"型,但CFC组细胞的凋亡率显著高于GFC组(P0.01);CFC组细胞中Dnmt1(P0.01)、Dnmt3b(P0.01)、Tet1(P0.05)、Tet2(P0.05)、H19(P0.05)和IGF2(P0.01)基因表达水平均显著低于GFC组,而Tet3、Dnmt3a和IGF2R在2组间无显著性差异;CFC组细胞中IGF2两个差异甲基化区域(DMR1和DMR2)的甲基化水平均显著低于GFC组(74.1%vs.57.8%,P0.01;76.8%vs.40.0%,P0.01),但IGF2-H19印记基因控制区域甲基化水平显著高于GFC组(68.8%vs.84.0%,P0.01)。体细胞核移植通过影响Dnmt和Tet家族基因的表达引起IGF2-H19基因座甲基化的异常,导致基因印记紊乱,进而影响再克隆的效率。
[Abstract]:To study the effect of somatic cell nuclear transfer on the methylation of IGF2-H19 locus in cloned fibroblasts, we used milk goat ear fibroblasts (GFC) and cloned goat ear fibroblasts (experimental group) as experimental materials. At the fifth passage, cell growth curve was plotted by cell count, apoptosis was detected by flow cytometry, and the difference of gene expression was analyzed by real-time fluorescence quantitative PCR. The methylation level of differential methylation regions was analyzed by bisulfite sequencing. The results showed that the growth curves of GFC and CFC cells were typical "S" type. However, the apoptotic rate of CFC group was significantly higher than that of GFC group. The expression levels of Dnmt1P0.01Tnmt3bP0.01Tet1Tet1P0.05Tet2P0.05H19P0.05) and IGF2P0.01) were significantly lower than those of GFC group. There was no significant difference between Tet3a and IGF2R. The methylation level of DMR1 and DMR2 was significantly lower than that of GFC group (74.1vs.57.80.76. 8V s.40.0P0.01a), but the level of methylation in the control region of IGF2-H19 imprinting gene was significantly higher than that in GFC group (68.8vs.84.0V). Nuclear transfer causes abnormal methylation of IGF2-H19 loci by affecting the expression of Dnmt and Tet family genes. Lead to gene imprinting disorder, and then affect the efficiency of re-cloning.
【作者单位】：南京农业大学动物科技学院;
【基金】：国家自然科学基金(31301973)
【分类号】：S827

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