TARGETED INTEGRATION OF ADENOASSOCIATED VIRUS-DERIVED PLASMIDS IN TRANSFECTED HUMAN-CELLS

Citation
L. Pieroni et al., TARGETED INTEGRATION OF ADENOASSOCIATED VIRUS-DERIVED PLASMIDS IN TRANSFECTED HUMAN-CELLS, Virology (New York, N.Y. Print), 249(2), 1998, pp. 249-259
Citations number
54
Categorie Soggetti
Virology
ISSN journal
00426822
Volume
249
Issue
2
Year of publication
1998
Pages
249 - 259
Database
ISI
SICI code
0042-6822(1998)249:2<249:TIOAVP>2.0.ZU;2-A
Abstract
Adeno-associated virus (AAV) integrates its genomic DNA into a defined region of human chromosome 19 (AAVS1). The specificity of integration is dependent on the presence of the inverted terminal repeats (ITR) a nd on expression of the rep gene. To develop Vectors capable of target ing the insertion of a selected DNA sequence into a specific location of human chromosome, we determined whether the rep gene can mediate si te-specific integration when cloned outside of an ITR-flanked transgen e cassette. HeLa and Huh-7 cells were transfected with a plasmid conta ining the rep gene, as well as the green fluorescent protein (GFP) and neomycin (neo) resistance gene inserted between the ITRs of AAV. Sout hern blot analysis or individual clones detected Rep-mediated site-spe cific integration of the ITR-flanked DNA in 25% and 12% of the Hela an d Huh-7 clones, respectively. The localization of the GFP-Neo sequence on chromosome 19 also was confirmed by fluorescent in situ hybridizat ion analysis of the transfected HeLa clones. Sequence analysis of the [TR-AAVS1 junction of one of the transfected Huh-7 clones indicated th at the insertion of the ITR DNA fragment had occurred at nucleotide 10 03. These results have implications for the development of AAV-derived Vectors capable of directing the site-specific integration of a gene of interest (C) 1998 Academic Press.