A 16 bp Rep Binding Element is Sufficient for Mediating Rep-dependent Integration into AAVS1

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• 作者：DengMin Feng ; JinZhong Chen ; YangBo Yue ; HuanZhang Zhu ; JingLun Xue and William WeiGuo Jia
• 刊名：Journal of Molecular Biology
• 出版年：2006
• 出版时间：21 April 2006
• 年：2006
• 卷：358
• 期：1
• 页码：38-45
• 全文大小：462 K

文摘

Adeno-associated virus (AAV) is a non-pathogenic virus and the only known eukaryotic virus capable of targeting human chromosome 19 for integration at a well-characterized AAVS1 site. Its site-specific integration is mediated by Rep68 and Rep78, viral proteins that bind to both the viral genome and AAVS1 site on ch19 through a specific Rep-binding element (RBE) located in both the viral genome and AAVS1. There are three RBEs in the AAV genome: two identical ones in both inverted terminal repeats (ITR) and another one in a recently discovered region termed the P5 integration efficiency element (P5IEE) that encompasses the viral P5 promoter. In order to identify the viral cis-acting sequence essential for Rep-mediated integration, we tested a series of constructs containing various lengths of P5IEE and compared the two RBEs from ITR (RBE_itr) and P5IEE (RBE_p5) in terms of their efficiency in Rep-dependent integration. Methods employed included a colony-forming assay, a PCR-based assay and Southern blotting analysis. We found that 16 bp of the RBE cis-element was sufficient for mediating Rep-dependent site-specific integration. Furthermore, RBE_itr was both more effective and specific than the RBE_p5 in Rep-dependent integration at the AAVS1 site. These findings added new information on the mechanism of Rep-dependent AAV genome insertion at the AAVS1 site and may be helpful in developing new high efficiency vectors for site-specific transgene integration.