Targeted transgene insertion into human chromosomes by adeno-associated virus vectors.
Efficient methods are needed for the precise genetic manipulation of diploid human cells, in which cellular senescence and low conventional gene targeting rates limit experimental and therapeutic options. We have shown previously that linear, single-stranded DNA vectors based on adeno-associated virus (AAV) could accurately introduce small (<20 bp) genetic modifications into homologous human chromosomal sequences. Here we have used AAV vectors to introduce large (>1 kb) functional transgene cassettes into the hypoxanthine phosphoribosyl transferase (HPRT) and Type I collagen (COL1A1) loci in normal human fibroblasts. The transgene cassettes are inserted at high frequencies (1% of the total cell population under optimal conditions) and without secondary mutations. Selection for the inserted transgene cassette can be used to enrich for targeting events, such that >70% of surviving cells have undergone gene targeting with an appropriately designed vector. This approach should prove useful both for functional genomic analysis in diploid human cells and for therapeutic gene targeting.
Hirata
Roli
R
Department of Medicine, Mailstop 357720, University of Washington, Seattle, WA 98195,, USA.
Chamberlain
Joel
J
Dong
Rong
R
Russell
David W
DW
D002478:Cells, Cultured; D002877:Chromosomes, Human; D024042:Collagen Type I / Q000235:genetics; D000229:Dependovirus / Q000235:genetics* / Q000276:immunology; D005347:Fibroblasts / Q000276:immunology / Q000502:physiology; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods; D019076:Transgenes
D024042:Collagen Type I; D007041:Hypoxanthine Phosphoribosyltransferase