核基质结合区的位置对转基因表达的影响  

Positional Effects of Matrix Attachment Regions on Transgene Expressions

作　　者：张俊河[1] 昝玉玺[1] 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

出　　处：《中国生物化学与分子生物学报》2009年第9期839-843,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目(No.30470030);河南省科技厅科技攻关项目(No.0624410041);河南省教育厅自然科学基金资助项目(No.2008A310008)~~

摘　　要：为研究核基质结合区(MAR)序列不同插入位置对转基因表达作用的影响,PCR扩增人β-珠蛋白MAR分别插入到含氯霉素乙酰转移酶(chloramphenicol acetyltransferase,CAT)报告基因真核表达载体pCATG表达盒两侧、5′端及3′端.酶切鉴定后,用阳离子聚合物转染CHO细胞,G418筛选出阳性细胞克隆,ELISA分析CAT基因的表达水平,半定量PCR分析CAT基因相对拷贝数.结果表明,表达盒两侧含MAR序列的载体能提高介导的转基因表达水平平均提高10.4倍,5′端含MAR序列的载体表达水平平均提高3.9倍,3′端含MAR序列的载体反而降低转基因表达水平.5′端含MAR序列的表达载体其转基因相对拷贝数高于其它两组载体的基因拷贝数,转基因表达量与基因拷贝数不成正比.To study the positional effect of matrix attachment regions（MARs）on the transgene expressions,PCR-amplified human β-globin MAR was inserted at different sites of a chloramphenicol acetyltransferase（CAT）-containing pCATG reporter vector,including a construct of CAT cassette flanked by two MARs,or a single MAR at either 5 or 3 .After transfected into CHO cells with positive ion liposomes and G418-screening,the CAT expression levels were determined by ELISA.The relatively CAT gene copies were determined by ELISA. The relatively CAT gene copies were determined by semi-quantitative PCR. The results showed that the CAT expression was increased most in two MARs flanked cassette. The 5＇-inserted MAR also increased, but the 3＇ MAR insertion decreased the transgene expression. We also found that the transgene expression failed to be in direct proportion to gene copy numbers.

关 键 词：核基质结合区 插入位置 转基因表达 报告基因 

分 类 号：Q781[生物学—分子生物学]