人β-干扰素核基质附着区在CHO细胞中对转基因表达的调控  

Regulatory effect of human beta-interferon matrix attachment region on transgene expression in CHO cells

作　　者：昝玉玺[1] 王天云[1] 张俊河[1] 王俐[1] 

机构地区：[1]新乡医学院生物化学与分子生物学教研室,河南省新乡市453003

出　　处：《中国组织工程研究与临床康复》2008年第29期5623-5626,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30470030);河南省自然科学基金(0511042300);河南省科技攻关项目(0624410041)~~

摘　　要：背景:核基质附着区是染色质被限制酶消化后仍附着在核基质上的DNA序列,不仅可影响邻近内源基因的表达,还能克服转基因沉默,提高外源基因的转录与表达。目的:探讨在CHO细胞中β-干扰素核基质附着区对氯霉素乙酰转移酶转基因表达的影响。设计、时间及地点:开放性实验,于2006-10/2007-04在新乡医学院生物化学与分子生物学实验室及分子研究室完成。材料:CHO细胞株由中国典型培养物保藏中心提供。含氯霉素乙酰转移酶报告基因及G418筛选标记的质粒pCATG载体由本实验室构建。方法:将PCR扩增得到的人β-干扰素核基质附着区分别用SacI/KpnI及BamHI/SalI酶切,连接至用相应内切酶酶切的pCATG载体上,转化E.coliJM109,提取质粒行酶切电泳,将构建好的载体命名为pCAT-MAR,该载体含氯霉素乙酰转移酶报告基因表达盒及两侧的β-干扰素核基质附着区。分别用载体pCATG及pCAT-MAR转化CHO细胞,提取具有G418抗性的细胞株基因组DNA,PCR扩增氯霉素乙酰转移酶基因。主要观察指标:ELISA法分析氯霉素乙酰转移酶报告基因的表达。PCR法检测pCAT-MAR载体是否稳定整合在宿主基因组上。结果:①pCATG转化的CHO细胞共筛选出16个阳性细胞株,pCAT-MAR转化的CHO细胞筛选出17个阳性细胞株。β-干扰素核基质附着区可使氯霉素乙酰转移酶报告基因表达水平提高2.8倍,pCATG转化CHO细胞的变异系数为2.0650,pCAT-MAR转化CHO细胞的变异系数仅为0.8131。②稳定转化的细胞株基因组DNA均扩增出437bp目的片段,证明pCAT-MAR载体已经稳定整合到基因组上。结论:β-干扰素核基质附着区能提高转基因在CHO细胞的表达水平,并且可降低不同转化细胞株之间转基因表达的差异性。BACKGROUND： Matrix attachment region （MAR）, a DNA sequence, is still bound to the nuclear matrices after chromatin digested with restriction endonuclease, not only affects expression of endogenous gene, but also overcames transgenic silence and improves transcription and expression of exogenous gene. OBJECTIVE： To investigate the influence of β -interferon MAR of CHO ceils on the transgenic expression of chlorampbenicol acetyltransferase （CAT）. DESIGN, TIME AND SETTING： The opening experiment was performed at the Department of Biochemistry and Molecular Biology, Molecular Institute, Xinxiang Medical College from October 2006 to April 2007. MATERIALS： CHO cell lines were obtained from China Center for Type Culture Collection. The pCATG vector of CAT and G418 screening markers were constructed by this laboratory. METHODS： Human β -interferon MAR by PCR was digested with SacI/KpnI and BamHI/SalI, and was inserted into pCATG vector, which was propagated in Escherichia coli JM109, then extracted and purified followed by enzyme digestion analysis. Vector of CAT expression cassette and human β-interferon MAR by the two sides was successfully constructed, and christened as pCAT-MAR. Two methods were compared between CHO cells of pCATG transformation and CHO cells of pCATG-MAR transformation. After （3418 selecting, genome DNA of cell lines of （3418 was extracted, then primers for PCR to amplify the CAT target gene fragment was designed. MAIN OUTCOME MEASURES： The activity of CAT was analyzed by ELISA method. It was also tested to see if the pCATG-MAR was stably integrated into genomic DNA in the transfected cells. RESULTS： CHO cells of pCATG transformation was screened to have 16 strains of positive cell, and CHO cells of pCATG-MAR transformation was screened to have 17 strains of positive cell. Human β -interferon MAR could increase the CAT gene expression by 2.8 fold. The coefficient of variation of CHO cells of pCATG transformation was 2.065 0, and coefficient of variation of CHO

关 键 词：核基质结合区 转基因 基因沉默 报告基因 

分 类 号：R394.2[医药卫生—医学遗传学]