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 sequences1,
2,
3,
4. 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.
