Targeted integration of adeno-associated virus-derived plasmids in transfected human cells.
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) and on expression of the rep gene. To develop vectors capable of targeting the insertion of a selected DNA sequence into a specific location of human chromosome, we determined whether the rep gene can mediate site-specific integration when cloned outside of an ITR-flanked transgene cassette. HeLa and Huh-7 cells were transfected with a plasmid containing the rep gene, as well as the green fluorescent protein (GFP) and neomycin (neo) resistance gene inserted between the ITRs of AAV. Southern blot analysis of individual clones detected Rep-mediated site-specific integration of the ITR-flanked DNA in 25% and 12% of the HeLa and Huh-7 clones, respectively. The localization of the GFP-Neo sequence on chromosome 19 also was confirmed by fluorescent in situ hybridization analysis of the transfected HeLa clones. Sequence analysis of the ITR-AAVS1 junction of one of the transfected Huh-7 clones indicated that the insertion of the ITR DNA fragment had occurred at nucleotide 1003. These results have implications for the development of AAV-derived vectors capable of directing the site-specific integration of a gene of interest.
Pieroni
L
L
Istituto di Ricerche di Biologia Molecolare "Piero Angeletti," P. Angeletti, Via Pontina Km 30,600, Pomezia, 00040, Italy.
Fipaldini
C
C
Monciotti
A
A
Cimini
D
D
Sgura
A
A
Fattori
E
E
Epifano
O
O
Cortese
R
R
Palombo
F
F
La Monica
N
N
D001483:Base Sequence; D002460:Cell Line; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D014162:Transfection; D016662:Virus Integration / Q000235:genetics*
D017931:DNA Primers; D004279:DNA, Viral