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Published online 2 January 2004

Nucleic Acids Research, 2004, Vol. 32, No. 1 e3
© 2004 Oxford University Press

Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses

Manu Kohli, Carlo Rago, Christoph Lengauer, Kenneth W. Kinzler and Bert Vogelstein*

The Howard Hughes Medical Institute, The Sidney Kimmel Comprehensive Cancer Center, and The Cellular and Molecular Medicine Program, The Johns Hopkins University Medical Institutions, Baltimore, MD 21231, USA

*To whom correspondence should be addressed. Email: vogelbe{at}welch.jhu.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

Emerging evidence suggests that recombinant adeno-associated viral (rAAV) vectors can be used for specific gene targeting in human somatic cells. We have developed an rAAV vector construction procedure employing fusion PCR and a single cloning step that considerably simplifies the knockout process. We demonstrate its utility by disrupting genes at specific positions within human colon cancer cells as well as within immortalized normal epithelial cells. This technology should be broadly applicable to in vitro studies that require the manipulation of the human genome.


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