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A 16bp Rep binding element is sufficient for mediating Rep-dependent integration into AAVS1.

DengMin Feng·JinZhong Chen·YangBo Yue·HuanZhang Zhu·JingLun Xue·William WeiGuo Jia
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Abstract

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 16bp 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.

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Base PairingBase SequenceCells, CulturedChromosomes, Human, Pair 19DNADNA-Binding ProteinsDependovirusHumansMolecular Sequence DataPlasmidsRegulatory Sequences, Nucleic AcidSequence AlignmentSequence DeletionViral ProteinsVirus IntegrationJournal ArticleResearch Support, Non-U.S. Gov't

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DengMin Feng, JinZhong Chen, YangBo Yue, HuanZhang Zhu, JingLun Xue, William WeiGuo Jia. 2006-01-30. A 16bp Rep binding element is sufficient for mediating Rep-dependent integration into AAVS1.. https://doi.org/10.1016/j.jmb.2006.01.029

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