Epitope tagging of endogenous genes in diverse human cell lines.
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.
Kim
Jung-Sik
JS
Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, DC 20057, USA.
Bonifant
Challice
C
Bunz
Fred
F
Lane
William S
WS
Waldman
Todd
T
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
D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; C085706:FLAG peptide; D051059:PTEN Phosphohydrolase
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