AAV-mediated gene targeting methods for human cells

作者：Khan Iram F; Hirata Roli K; Russell David W^*

来源：Nature Protocols, 2011, 6(4): 482-501.

DOI：10.1038/nprot.2011.301

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 similar to 10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks.

• 出版日期2011-4