A 16bp Rep binding element is sufficient for mediating Rep-dependent integration into AAVS1.
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.
Feng
DengMin
D
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China.
Chen
JinZhong
J
Yue
YangBo
Y
Zhu
HuanZhang
H
Xue
JingLun
J
Jia
William WeiGuo
WW
D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics*; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D014764:Viral Proteins / Q000378:metabolism*; D016662:Virus Integration / Q000235:genetics*
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D004247:DNA