Nucleic Acids Research Advance Access originally published online on September 10, 2008
Nucleic Acids Research 2008 36(19):e127; doi:10.1093/nar/gkn566
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Nucleic Acids Research, 2008, Vol. 36, No. 19 e127
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Epitope tagging of endogenous genes in diverse human cell lines
1Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, DC 20057, 2Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21231 and 3FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138, USA
*To whom correspondence should be addressed. Tel: +1 202 6871340; Fax: +1 202 6877505; Email: waldmant{at}georgetown.edu
Received April 1, 2008. Revised August 21, 2008. Accepted August 21, 2008.
Epitope tagging is a powerful and commonly used approach for studying the physical properties of proteins and their functions and localization in eukaryotic cells. In the case of Saccharomyces cerevisiae, it has been possible to exploit the high efficiency of homologous recombination to tag proteins by modifying their endogenous genes, making it possible to tag virtually every endogenous gene and perform genome-wide proteomics experiments. However, due to the relative inefficiency of homologous recombination in cultured human cells, epitope-tagging approaches have been limited to ectopically expressed transgenes, with the attendant limitations of their nonphysiological transcriptional regulation and levels of expression. To overcome this limitation, a modification and extension of adeno-associated virus-mediated human somatic cell gene targeting technology is described that makes it possible to simply and easily create an endogenous epitope tag in the same way that it is possible to knock out a gene. Using this approach, we have created and validated human cell lines with epitope-tagged alleles of two cancer-related genes in a variety of untransformed and transformed human cell lines. This straightforward approach makes it possible to study the physical and biological properties of endogenous proteins in human cells without the need for specialized antibodies for individual proteins of interest.