A method mediated AAVS1 recombination with Rep mRNA and homologous arms  

作　　者：Qiantong Xiang Linian Huang Sijia Guo Fang Chen Xiaojun Zha Bing Chen Liangdan Sun Haisheng Zhou Depei Liu 

机构地区：[1]Department of Biochemistry and Molecular Biology,Anhui Medical University,Hefei 230032,China [2]Department of Respiratory Diseases,Bengbu Medical College,Bengbu 233001,China [3]Key Laboratory of Dermatology(Anhui Medical University),Ministry of Education,Hefei 230032,China [4]National Laboratory of Medical Molecular Biology,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100005,China

出　　处：《Acta Biochimica et Biophysica Sinica》2012年第12期1015-1022,共8页生物化学与生物物理学报（英文版）

基　　金：supported by the grants from the General Program of National Natural Science Foundation of China(81172591);Anhui Provincial Natural Science Foundation(090413074);the Open Funds of the State Key Laboratory of Medical Molecular Biology(Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences)(2060204);Scientific Research of BSKY(XJ200801)from Anhui Medical University.

摘　　要：The adeno-associated virus （AAV） genome can be stably integrated into the AAVS1 region of human chromosome 19 （19q13.4-qter） with the assistance of Rep68/78 protein. In the current models of AAV integration in a locus- specific manner, the foreign genes were randomly inserted into the AAVS1 region, which contains several functional genes. As random integration in this region may lead to insertion mutations and disrupt normal gene expression or critical signaling pathways of the host cells, it is neces- sary to find a precise insertion site in the AAVS1 region. Homologous recombination is the most accurate and ver- satile mechanism for such site-specific integration. To in- vestigate site-specific integration in the AAVS1 region, a targeted vector containing two homologous arms derived from AAVS1 and a reporter gene was transfected into HeLa cells with or without Rep68/78 mRNA. The results indicated that transient expression of Rep68/78 in HeLa cells improved integration of the gene of interest at the AAVS1 locus in a site-specific manner. Compared with locus-specific integration reported in previous studies, site- specific integration may minimize the risk associated with random DNA integration in the AAVS1 region, which might be helpful for gene therapy.The adeno-associated virus （AAV） genome can be stably integrated into the AAVS1 region of human chromosome 19 （19q13.4-qter） with the assistance of Rep68/78 protein. In the current models of AAV integration in a locus- specific manner, the foreign genes were randomly inserted into the AAVS1 region, which contains several functional genes. As random integration in this region may lead to insertion mutations and disrupt normal gene expression or critical signaling pathways of the host cells, it is neces- sary to find a precise insertion site in the AAVS1 region. Homologous recombination is the most accurate and ver- satile mechanism for such site-specific integration. To in- vestigate site-specific integration in the AAVS1 region, a targeted vector containing two homologous arms derived from AAVS1 and a reporter gene was transfected into HeLa cells with or without Rep68/78 mRNA. The results indicated that transient expression of Rep68/78 in HeLa cells improved integration of the gene of interest at the AAVS1 locus in a site-specific manner. Compared with locus-specific integration reported in previous studies, site- specific integration may minimize the risk associated with random DNA integration in the AAVS1 region, which might be helpful for gene therapy.

关 键 词：adeno-associated virus homologous recombination AAVS 1 Rep68/78 locus-specific integration site-specific integration 

分 类 号：Q78[生物学—分子生物学] Q255