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
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.