绝缘子元件的克隆及功能验证  

Cloning of Insulator Element and Testification of its Function

作　　者：王延安[1] 张志愿[1] 费照亮 蒋欣泉 王铸钢 郑家伟[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院口腔颌面外科,上海200011 [2]上海南方模式生物研究中心,上海200015 [3]上海市口腔医学研究所,上海200011

出　　处：《口腔颌面外科杂志》2007年第4期297-300,共4页Journal of Oral and Maxillofacial Surgery

基　　金：国家自然科学基金(30371546);上海市重点学科(优势学科)建设项目(Y0203)

摘　　要：目的:克隆绝缘子元件并验证其绝缘功能,为转基因载体的构建提供条件。方法:以GenBank提供的绝缘子序列设计引物,应用PCR方法,从鸡的基因组序列中克隆绝缘子,酶切、测序鉴定;将绝缘子亚克隆至表达载体pEGFP-N2,体外扩增纯化后瞬时转染COS7细胞,72h后收获细胞,荧光显微镜下观察EGFP表达情况。结果:绝缘子元件成功克隆,酶切、测序鉴定序列正确;体外试验证实,亚克隆有绝缘子的载体与对照组比较,EGFP表达水平显著降低。结论:绝缘子元件成功克隆,且在体外具有绝缘功能,能够与目的基因共同构建转基因载体,从而提高转基因的表达效率。Objective： To clone the cHS4 insulator and testify its function in vitro. Methods： The genomic DNA was isolated from chicken, and cHS4 insulator was cloned by PCR method.The element was testifid through enzyme digestion and gene sequencing. The insulator element was subcloned into the vector of pEGFP-N2, and constructed plasmid pN2- hs4EGFP. The plasmid was transfected into COS7 cells to testified the function of insulator. After 72 hours of incubation, the cells were observed under fluorescent light. Results： The insulator was cloned successfully, and the expression of EGFP in the plasmid of pN2-hs4EGFP was much lower than that of positive control group. Conclusion： The insulator was cloned successfully, and worked well in vitro. This element may be used in the construction of transgenic vectors,so as to improve the expression of the interest genes.

关 键 词：绝缘子 转基因 绿色荧光蛋白 瞬时转染 转基因沉默 

分 类 号：Q78[生物学—分子生物学]