AAV-mediated gene targeting methods for human cells

Abstract

Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10−5 to 10−2 per infected cell. these targeting frequencies are 1–4 logs higher than those obtained by conventional transfection or electroporation approaches. a wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by southern blots. this protocol (from vector design through a single round of targeting and screening) can be completed in ~10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks.

Sister chromatid telomere fusions, but not NHEJ-mediated inter-chromosomal telomere fusions, occur independently of DNA ligases 3 and 4

“…They were generated using recombinant adenoviral-associated (rAAV) and CRISPR/Cas9 (LIG3 −/− :LIG4 −/− line only) targeting methods, as described (Khan et al 2011;Oh et al 2014;B Ruis, T Takasugi, S Oh, EA Hendrickson, unpubl.). These cell lines represent our models of DNA repair pathways that are selectively compromised.…”

RNA interference of influenza A virus replication by microRNA-adapted lentiviral loop short hairpin RNA

“…Therefore, screening the monoclonal shRNAmir-expressing cells may result in more efficient miRNA expression and targeted knockdown. Alternatively, targeted integration at a defined locus such as the AAVS1 locus (safe harbour site) may also improve miRNA expression (DeKelver et al, 2010;Khan et al, 2011). We also found that expression levels of miRNAs in tandem-shRNAmir-transduced 293T and MDCK cells were different (Fig.…”

The Role of DNA Repair Pathways in Adeno-Associated Virus Infection and Viral Genome Replication / Recombination / Integration

“…In this regard, rAAV has become increasingly popular as the most efficient tool to precisely introduce defined DNA modifications at the target site in the cellular genome with remarkably high efficiencies of up to 1% in the cell population (Hendrie & Russell, 2005;Khan et al, 2011;Russell & Hirata, 1998;Vasileva & Jessberger, 2005). Targeting efficiencies could be increased further by 60-100 fold or more by introducing a DSB at the target site with a site-specific endonuclease (Miller et al, 2003;Porteus et al, 2003).…”