转基因下游MAR在稳定转化的CHO细胞中对基因表达的调控作用  

Regulatory Effect of Transgenic Downstream MAR on Gene Expression in Stable Transfected CHO Cells

作　　者：张俊河[1] 张艳芳[1] 王芳[1] 杨献军[2] 王天云[1] 

机构地区：[1]新乡医学院生物化学与分子生物学教研室,河南新乡453003 [2]新乡医学院分子生物学研究室,河南新乡453003

出　　处：《安徽农业科学》2009年第18期8359-8361,共3页Journal of Anhui Agricultural Sciences

基　　金：河南省科技厅科技攻关项目(0624410041);河南省教育厅自然科学基金(2008A310008)资助

摘　　要：[目的]探讨转基因下游MAR在稳定转化的CHO细胞中对基因表达的调控作用。[方法]将PCR扩增得到的人β-珠蛋白MAR插入到真核表达载体pCATG的下游,构建转基因表达盒3′端含MAR的表达载体。酶切鉴定正确后,转染CHO细胞,G418筛选出稳定转化的细胞株,ELISA分析转基因的表达水平,半定量PCR分析转基因相对拷贝数。[结果]结果表明,表达盒3′端含MAR序列能使转基因表达水平降低,其基因拷贝数则有一定程度的增加。转基因下游β-珠蛋白MAR在一定程度上能抑制外源基因的表达水平;外源基因表达量与基因拷贝数不成正比,未呈现出"拷贝数依赖性"。[结论]该研究结果为进一步研究MAR的调控机制奠定基础。[Objective] The purpose of this research was to investigate the influence of transgcnic downstream human β-globin matrix attachment region （MAR）on gene expression in stable transfected CHO cells. [ Method ] Human β-globin MAR was amplified through PCR, and was inserted into 3＇site of pCATG vector to construct the expression vector which contained the β-globin MAR at 3 ＇site of Chloramphenicol acetyl-transferase （CAT） reporter gene expression cassette. After the identification of enzyme digestion, CHO ceils were transfected. The CAT gene expression was analyzed by ELISA methods after screening by G418. The relatively CAT gene copy numbers were determined through semiquantitative PCR. [Result] The results indicated that the expression vector which contained MAR at 3＇site decreased the CAT gene expression level, while its CAT gene copy numbers of the ceils transformed with the expression vector were higher than the control vector. Transgenic downstream human β-globin MAR could inhibit foreign gene expression to some extent. The expression level of foreign gene was not in direct proportion to gene copy numbers, gene copy numbers dependent did not exist. [Conclusion] This research will lay the foundation for further study of regulatory mechanism of MAR.

关 键 词：核基质结合区 CHO细胞 转基因 报告基因 

分 类 号：Q782[生物学—分子生物学]