中文摘要：

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。

英文摘要：

The pCMV-EGFP gene was amplified by PCR using pEGFP-NI 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 technique and PCR. Results showed that the recombinant expression vector pMD19-DS2-pCMV-EGFP-DS1 was constructed and transfected into HEK293 cells successfully. The green fluorescence of EGFP was sustained and stable in transfected cells. The efficiency of the homogenous recombination was up to 4%. Using the target of multiple sites improved the high efficiency of gene targeting without selection. It was more than 4000 folds of the general gene targeting. The gene targeting using the recombinant vector has also partially solved the problems of unable expression of inserted genes and has solved the need for safety against toxicity integration.