克隆山羊成纤维细胞IGF2-H19基因座甲基化分析  

IGF2-H19 Locus Methylation Status in Cloned Goat Fibroblast Cells

作　　者：刘孜斐 邓明田 任才芳[1] 万永杰[1] 王锋[1] 

机构地区：[1]南京农业大学动物科技学院,南京210095

出　　处：《畜牧兽医学报》2017年第12期2277-2285,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31301973)

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(P<0.01);CFC组细胞中Dnmt1(P<0.01)、Dnmt3b(P<0.01)、Tet1(P<0.05)、Tet2(P<0.05)、H19(P<0.05)和IGF2(P<0.01)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%,P<0.01;76.8%vs.40.0%,P<0.01),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%,P<0.01)。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。The aim of this experiment was to study the influence of somatic cell nuclear transfer on IGF2-H19 locus methylation in cloned goat fibroblast cells. Goat ear fibroblast cells （GFC, control group） and cloned goat ear fibroblast cells （CFC, experimental group） were used as experimental materials. When cells in the 2 groups were cultured to passage 5, using cell counting method for cell growth curves, flow cytometer for cell apoptosis, real-time PCR for gene relative expression level and bisulfite sequencing PCR for the methylation status of differentially methylated region （DMR）. The results showed that cell growth curves in the 2 groups were typical ＂S＂-shaped, but apoptosis rate in CFC group was increased significantly（P〈0.01） compared with that in GFC group. The expression level of Dnmt1 （P〈0.01）, Dnmt3b （P〈0.01）, Tet1 （P〈0.05）, Tet2 （P〈0.05）, H19 （P〈0.05） and IGF2 （P〈0.01） in CFC group were decreased significantly compared with that in GFC group. However, no statistical differences were found at the expression levels of Tet3、Dnmt3a and IGF2R between the 2 groups. Compared with GFC group, the methylation level of IGF2 DMR1 （74.1% vs. 57.8%, P〈0.01） and DMR2 （76.8% vs. 40.0%, P〈0.01） were decreased significantly, while the methylation level of IGF2-H19 imprinting control region （ICR） was increased significantly （68.8% vs. 84.0%, P〈0.01） in CFC group. Somatic cell nuclear transfer resulted in aberrant methylation of IGF2-H19 locus by influencing Dnmts and Tets genes expression, which could affect genomic imprinting, and then influence the efficiency of serial recloning.

关 键 词：体细胞核移植 再克隆 IGF2-H19 DNA甲基化 山羊耳成纤维细胞 

分 类 号：S827.2[农业科学—畜牧学]