转基因猪细胞中CMV启动子区域甲基化的研究  

The Methylation Level of CMV in Fibroblasts of Transgenic Pigs

作　　者：任广彩[1,2] 张英[1] 丛佩清[1] 陈瑶生[1] 何祖勇[1] 

机构地区：[1]中山大学生命科学学院∥有害生物控制与资源利用国家重点实验室,广东广州510006 [2]广东大华农动物保健品股份有限公司,广东广州510642

出　　处：《中山大学学报（自然科学版）》2015年第6期14-18,共5页Acta Scientiarum Naturalium Universitatis Sunyatseni

基　　金：国家转基因重大专项资助项目(2011ZX08006-005)

摘　　要：为研究转基因猪外源基因表达与启动子区域甲基化的关系,利用流式细胞仪检测体外传代至第5、10和15代的转基因猪成纤维细胞中EGFP荧光强度的变化情况;采用亚硫酸盐测序的方法对这3代细胞CMV IE启动子区域30个Cp G岛的甲基化程度进行测定。结果显示细胞从第5代培养到第10代时荧光变弱,但是当继续培养到15代时EGFP荧光又渐渐变强,但EGFP阳性细胞的比例没有发生变化,均为100%。第5、10、15代细胞启动子区域的Cp G岛甲基化阳性率分别为3.64%±1.25、2.97%±1.03和1.65%±0.74,统计结果表明3代细胞间的甲基化阳性率差异不显著(P>0.05)。表明转基猪成纤维细胞中CMV启动子区域的甲基化程度随着体外培养时间的变化不明显,与EGFP的表达水平变化无明显相关。To investigate the relationship between the expression levels of exogenous gene and methylation level of promoter in cells derived from transgenic pigs,the EGFP fluorescence of the fifth,10^th and15 thgeneration fibroblast of EGFP transgenic pigs were detected by flow cytometry,and the methylation status of a 560-bp region of the CMV promoter containing one Cp G island including 30 Cp G sites were examined by bisulfite sequencing method. The mean fluorescence intensity declined from the fifth generation of transgenic pig cells to the 10^th generation cells,however,it increased to the level comparable to the fifth generation of cells from the 10^th to 15th cells. The methylation level of the fifth,10^th and 15th generation cells of the CMV promoter were 3. 64% ± 1. 25,2. 97% ± 1. 03 and 1. 65% ± 0. 74,respectively. The difference of methylation level among different generation of transgenic cells was insignificant,which indicated that the expression level of exogenous gene EGFP was not closely correlated to the methylation level on CMV promoter.

关 键 词：甲基化 启动子 CPG岛 转基因猪 

分 类 号：S828[农业科学—畜牧学] Q78[农业科学—畜牧兽医]