富含GC的DNA片段对中国仓鼠卵巢细胞中转基因表达的影响及其位置效应  

Effect of GC-rich DNA fragment on the level of transgene expression in Chinese hamster ovary cells and its position effect

作　　者：陈思佳[1] 赵春澎[1] 张俊河[1] 王小引[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：《新乡医学院学报》2017年第9期794-797,共4页Journal of Xinxiang Medical University

基　　金：国家自然科学基金资助项目(编号:81673337)

摘　　要：目的分析富含GC的DNA片段对中国仓鼠卵巢(CHO)细胞中转基因表达水平的影响及其位置效应。方法人工合成富含GC的DNA片段,将其克隆到表达载体的表达盒的5'、3'端及两端,构建在载体不同位置插入富含GC DNA片段的3个新表达载体(p IRES-G1、p IRES-G2和p IRES-G3),分别转染CHO细胞并用荧光显微镜进行观察;对照载体为p IRES-EGFP。采用G418筛选稳定细胞株,流式细胞术分析增强型绿色荧光蛋白(EGFP)基因表达,实时荧光定量聚合酶链式反应(qRT-PCR)检测其转基因拷贝数。结果成功构建了在载体不同位置含有GC DNA片段的3个载体,瞬时及稳定转染结果显示,在载体表达盒两端及3'端插入富含GC DNA片段能够明显提高EGFP在CHO细胞中的表达水平。与对照载体比较,稳定表达情况下载体表达盒两端及3'端插入富含GC DNA能分别提高转基因表达水平约1.39、1.32倍,而在5'端插入富含GC DNA片断对于提高基因表达水平作用尚不明显。表达盒两端及3'端插入富含GC DNA片段的转基因拷贝数比对照载体提高了约1.32、1.24倍。结论富含GC的DNA片段在CHO细胞提高转基因表达与其在载体上的位置有关,在载体表达盒两端或3'端插入一段富含GC的DNA片段可以提高转基因表达水平。Objective To analyze the effect of GC-rich DNA fragments on the level of transgenic expression in Chinese hamster ovary （CHO） celts and its position effect.Methods The synthetic DNA fragment with GC-rich was cloned into the 5＇or 3＇or both 5＇and 3＇ends of expression cassette of expression vector.Three new expression vectors （pIRES-G1,pIRES-G2 and pIRES-G3） which was inserted with the GC-rich DNA fragments in different position were transfected CHO ceils,respectively,and then was observed under fluorescence microscope;the control vector was pIRES-EGFP.Stable transfected cell lines were screened under G418,and enhanced green fluorescent protein（EGFP） expression was analyzed by flow cytometry and the transgenic copy number was detected by quantitative real-time quantitative polymerase chain reaction （qRT-PCR）.Results Three expression vectors with a GC-rich DNA fragments in different position were constructed successfully.The insertion of GC-rich DNA fragments at 3＇end and both 5＇,3＇ends of the box of expression vector could obviously improve the expression level of vector in CHO cells;and the expression level of the stably transfected CHO cells increased 1.39 fold and 1.32 fold compared to the control vector,respectively;the transgene copy number increased 1.32 fold and 1.24 fold compared with the control vector.While the insertion of GC-rich DNA fragments at 5＇end of expression cassette had no obvious effect on the level of gene expression.Conclusion The role of DNA fragment with GC-rich in improving the transgenic expression of CHO cells is related to its position in the vector.The insertion of GC-rich DNA fragments at 3＇end and both 5＇,3＇ends of the box of expression vector can improve transgenic expression.

关 键 词：富含GC的DNA片段 转基因 位置效应 

分 类 号：Q7[生物学—分子生物学]