一个用于转染细胞分选的双顺反子载体的构建及其应用  

A Bicistronic Expression Vector for The Selection of Transfected Cells

作　　者：曲笑霞[1] 秦立蓬[1] 岳文[1] 高艳红[1] 李艳华[1] 袁红丰[1] 王韫芳[1] 刘大庆[1] 闫舫[1] 施双双[1] 裴雪涛[1] 

机构地区：[1]军事医学科学院输血研究所,干细胞研究室,北京100850

出　　处：《生物化学与生物物理进展》2005年第9期889-894,共6页Progress In Biochemistry and Biophysics

基　　金：国家高技术"863"计划资助项目(2002AA205050和2003AA205160);国家重点基础研究发展规划项目(973)(2001CB509906);北京市科委自然科学基金资助项目(H020220010190).~~

摘　　要：为简化转染细胞的分选过程,构建了一个含有细胞表面标志CD34基因的双顺反子载体p3.1-IRES-CD34.利用来源于脑心肌炎病毒(EMCV)的内部核糖体进入位点(IRES),实现目的基因与CD34基因的共同表达.将绿色荧光蛋白(EGFP)作为目的基因插入载体的多克隆位点,然后转染NIH-3T3细胞,通过免疫磁珠分选(MACS)方法来分选细胞.结果表明:对于转染细胞,均可实现快速分选(瞬时转染细胞约48h,稳定转染10￣15天),并且获得较高纯度(95%以上)的表达目的基因细胞.A bicistronic expression vector, p3.1-IRES-CD34, has been constructed to facilitate the selection and screening of transfected cells by magnetic cell sorting （MACS）. In this vector, an engineered variant of CD34, deleted CD34 （dCD34）, was chosen as the marker gene and the internal ribosome entry site （IRES） from encephalomyocarditis virus （EMCV） was employed to allow simultaneous expression of the inserted 5＇-end heterologous gene and the CD34 marker gene. To test the utility of the vector, the enhanced green fluorescent protein （EGFP） gene was inserted into the multiple cloning site （MCS） of the vector. Transfected cells were selected by magnetic cell sorting （MACS）. The results demonstrated that the transfected cells can be enriched to high purity （〉95%）. The vector p3.1-IRES-CD34 would provide a rapid （2～3 days for transient transfected cells and 10 - 15 days for stable transfected cells） and efficient method for the selection oftransfected cells.

关 键 词：载体 转染细胞 分选 CD34 

分 类 号：Q78[生物学—分子生物学]