Published online 7 October 2005
Methods Online |
Improved methods for the generation of human gene knockout and knockin cell lines
Department of Radiation Oncology and Molecular Radiation Sciences, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine Baltimore, MD 21231, USA 1Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine Baltimore, MD 21231, USA 2Department of Pediatrics, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine Baltimore, MD 21231, USA
*To whom correspondence should be addressed. Tel: +1 410 502 7941; Fax: +1 410 502 2821; Email: fbunz{at}jhmi.edu
Received August 11, 2005. Revised September 26, 2005. Accepted September 26, 2005.
Recent studies have demonstrated the utility of recombinant adeno-associated viral (rAAV) vectors in the generation of human knockout cell lines. The efficiency with which such cell lines can be generated using rAAV, in comparison with more extensively described plasmid-based approaches, has not been directly tested. In this report, we demonstrate that targeting constructs delivered by rAAV vectors were nearly 25-fold more efficient than transfected plasmids that target the same exon. In addition, we describe a novel vector configuration which we term the synthetic exon promoter trap (SEPT). This targeting element further improved the efficiency of knockout generation and uniquely facilitated the generation of knockin alterations. An rAAV-based SEPT targeting construct was used to transfer a mutant CTNNB1 allele, encoding an oncogenic form of ß-catenin, from one cell line to another. This versatile method was thus shown to facilitate the efficient integration of small, defined sequence alterations into the chromosomes of cultured human cells.
Present address: Ozlem Yildirim, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA
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