H. Tsunoda et al., Site-specific integration of adeno-associated virus-based plasmid vectors in lipofected HeLa cells, VIROLOGY, 268(2), 2000, pp. 391-401
Adeno-associated virus (AAV) integrates specifically into a site (AAVS1) on
human chromosome 19q13.3-qter. Similarly, there is accumulating evidence t
hat this site-specific integration occurs by transfection of AAV-based plas
mid vectors. In order to further define the process of plasmid integration
events, we constructed some AAV plasmids, introduced them into HeLa cells b
y lipofection, and isolated chromosomal integrants. One of such plasmids, p
TH-5, contained the rep and neomycin-resistant (neo(r)) genes flanked by th
e 5'- and 3'-inverted terminal repeats of AAV and the hygromycin-resistant
(hyg(r)) gene located in the plasmid backbone. Southern blot analysis revea
led that among 36 G418-resistant (G418') clones isolated, 22 (61%) showed s
ite-specific integration into AAVS1. Further structural and functional anal
yses on the expression of the hyg(r) gene in the site-specific clones and t
he LacZ gene in clones generated with plasmid pTH-2 indicated that, togethe
r with the AAV sequence, the plasmid backbone was integrated into the AAVS1
site and thus the neo(r) and hyg(r) genes remained linked at high frequenc
ies in the targeted integrants compared with random integrants. Sequence an
alysis of integration junctions between pTH-5 and AAVS1 revealed that the j
unctions occurred in the p5 promoter region of the plasmid while mainly in
the partial cDNA coding region of the AAVS1 site. We also found that plasmi
d pTH-1 linearized in the backbone before lipofection gave a significantly
lower frequency of site-specific integration (26%) than the circular form (
60%). This finding may support the involvement of the double-stranded, circ
ular form of infected AAV in the integration process. Our results may help
to understand the process and mechanism of site-specific integration of lip
ofected AAV plasmid vectors. (C) 2000 Academic Press.