Epitope tagging of endogenous genes in diverse human cell lines

Jung Sik Kim, Challice Bonifant, Fred Bunz, William S. Lane, Todd Waldman

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article numbere127
JournalNucleic Acids Research
Volume36
Issue number19
DOIs
StatePublished - 2008

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Epitopes
Cell Line
Genes
Homologous Recombination
Proteins
Dependovirus
Gene Knockout Techniques
Transformed Cell Line
Gene Targeting
Neoplasm Genes
Eukaryotic Cells
Transgenes
Proteomics
Saccharomyces cerevisiae
Cultured Cells
Alleles
Genome
Technology
Antibodies

ASJC Scopus subject areas

  • Genetics

Cite this

Epitope tagging of endogenous genes in diverse human cell lines. / Kim, Jung Sik; Bonifant, Challice; Bunz, Fred; Lane, William S.; Waldman, Todd.

In: Nucleic Acids Research, Vol. 36, No. 19, e127, 2008.

Research output: Contribution to journalArticle

Kim, Jung Sik ; Bonifant, Challice ; Bunz, Fred ; Lane, William S. ; Waldman, Todd. / Epitope tagging of endogenous genes in diverse human cell lines. In: Nucleic Acids Research. 2008 ; Vol. 36, No. 19.
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