| 多位点基因打靶的定点整合效率研究 |
| |
| 引用本文： | 王克振,蒋泓,唐冬生,曾芳,梁沂梅,李月琴,周天鸿.多位点基因打靶的定点整合效率研究[J].广东农业科学,2009(5):158-161. |
| |
| 作者姓名： | 王克振 蒋泓 唐冬生 曾芳 梁沂梅 李月琴 周天鸿 |
| |
| 作者单位： | 王克振,李月琴,周天鸿(暨南大学生命科学技术学院,广东广州,510632);蒋泓,唐冬生,曾芳,梁沂梅(佛山科学技术学院医学院,广东佛山,528000) |
| |
| 基金项目： | 国家自然科学基金项目，广东省科技攻关计划项目 |
| |
| 摘 要： | 利用PCR方法从pEGFP-N1中扩增pCMV-EGFP,从人基因组rDNA基因家族靶基因插入位点两侧分别扩增两条基因打靶同源重组引导序列DS1和DS2,将DS1、DS2片段插入pMD19-pCMV-EGFP中,构建成多位点基因打靶载体pMD19-DS2-pCMV-EGFP-DS1.通过脂质体将其转染至HEK293细胞内.通过荧光检测和PCR、测序等方法,检测和评价定点整合效率.试验结果表明,外源基因EGFP在转染细胞中持续稳定表达,EGFP定点整合率约为4%,不经药物筛选大大提高了整合效率,比传统的基因打靶技术提高了4000多倍,为转基因动物研究建立了高效的定点转基因技术.
|
| 关 键 词： | 基因打靶 多位点 定点整合 HEK293细胞 |
Research on the targeted integration efficiency of gene targeting of multiple sites |
| |
| WANG Ke-zhen,JIANG Hong,TANG Dong-sheng,ZENG Fang,LIANG Yi-mei,LI Yue-qin,ZHOU Tian-hong.Research on the targeted integration efficiency of gene targeting of multiple sites[J].Guangdong Agricultural Sciences,2009(5):158-161. |
| |
| Authors: | WANG Ke-zhen JIANG Hong TANG Dong-sheng ZENG Fang LIANG Yi-mei LI Yue-qin ZHOU Tian-hong |
| |
| Institution: | WANG Ke-zhen1,JIANG Hong2,TANG Dong-sheng2,ZENG Fang2,LIANG Yi-mei2,LI Yue-qin1,ZHOU Tian-hong1(1.College of Life Science and Technology,Jinan University,Guangzhou 510632,China 2.Medical College of Foshan University,Foshan 528000,China,) |
| |
| Abstract: | The pCMV-EGFP gene was amplified by PCR using pEGFP-N1 out of MCS as template.DS1 and DS2 were two homogenous recombination arms.Both of them were amplified from human genome by PCR.Each of them was cloned to the pMD19-T vector and the recombinant vector pMD19-DS2-pCMV-EGFP-DS1 was constructed.The plasmid was transfected into HEK293 cells using lipofectamine.Then the cultured HEK293 cells were gathered and used to detect and evaluate the efficiency of the recombination by using the fluorescent microscopy te... |
| |
| Keywords: | gene targeting multiple sites targeted integration HEK293 cells |
| 本文献已被 CNKI 万方数据 等数据库收录！ |
| 点击此处可从《广东农业科学》浏览原始摘要信息 |
| 点击此处可从《广东农业科学》下载免费的PDF全文 |
|
