title: Epitope tagging of endogenous genes in diverse human cell lines.
abstract: 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.
authors.affiliation: Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, DC 20057, USA.
authors.forename: Jung-Sik
authors.initials: JS
authors.lastname: Kim
authors.forename: Challice
authors.initials: C
authors.lastname: Bonifant
authors.forename: Fred
authors.initials: F
authors.lastname: Bunz
authors.forename: William S
authors.initials: WS
authors.lastname: Lane
authors.forename: Todd
authors.initials: T
authors.lastname: Waldman
mesh_terms: D000483:Alleles; D002460:Cell Line; D002461:Cell Line, Transformed; D000229:Dependovirus / Q000235:genetics; D000939:Epitopes / Q000032:analysis*; D018390:Gene Targeting / Q000379:methods*; D016158:Genes, p53; D005822:Genetic Vectors; D006801:Humans; D009842:Oligopeptides; D051059:PTEN Phosphohydrolase / Q000235:genetics; D010455:Peptides / Q000032:analysis* / Q000276:immunology
chemical_list: D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; C085706:FLAG peptide; D051059:PTEN Phosphohydrolase
references.citation: Mol Cell Biol. 2007 Jan;27(2):662-77
references.pmid: 17060456
references.citation: Nat Protoc. 2007;2(11):2734-46
references.pmid: 18007609
references.citation: Nucleic Acids Res. 2004 Jan 02;32(1):e3
references.pmid: 14704360
references.citation: Nature. 2006 Mar 30;440(7084):631-6
references.pmid: 16429126
references.citation: Cancer Res. 2004 Oct 1;64(19):6906-14
references.pmid: 15466180
references.citation: Biochemistry. 1998 Aug 4;37(31):10866-70
references.pmid: 9692979
references.citation: Nucleic Acids Res. 2005 Oct 07;33(18):e158
references.pmid: 16214806
references.citation: Nature. 2002 Apr 11;416(6881):648-53
references.pmid: 11923872
references.citation: Nat Methods. 2008 Feb;5(2):163-5
references.pmid: 18176569
references.citation: J Am Soc Mass Spectrom. 1994 Nov;5(11):976-89
references.pmid: 24226387
references.citation: Eur J Biochem. 1993 Dec 1;218(2):273-81
references.pmid: 7505743
references.citation: Nucleic Acids Res. 1983 Mar 11;11(5):1475-89
references.pmid: 6828386
references.citation: Mol Ther. 2002 Aug;6(2):272-8
references.pmid: 12349826
references.citation: Nat Biotechnol. 2002 Jul;20(7):735-8
references.pmid: 12089561
references.citation: Mol Cell Biol. 2003 May;23(10):3558-65
references.pmid: 12724414
references.citation: Mol Endocrinol. 2005 Oct;19(10):2451-65
references.pmid: 16051665