Epitope tagging of endogenous proteins for genome-wide chromatin immunoprecipitation analysis.
The development of chromatin immunoprecipitation methods coupled with DNA microarray (ChIP-chip) technology has enabled genome-wide identification of cis-DNA regulatory elements to which transcription factors bind. Nonetheless, the ChIP-chip technology requires antibodies with extremely high affinity and specificity for the target transcription factors. Unfortunately, such antibodies are not available for most human transcription factors. In principle, this problem can be circumvented by utilizing ectopically expressed epitope-tagged proteins recognizable by well-characterized antibodies. However, such expression is no longer endogenous. To surmount this problem, we have successfully developed a facile method to knock in a 3xFlag epitope into the endogenous gene loci of transcription factors. The knock-in approach provides a general solution for the study of proteins for which antibodies are substandard or not available.
Wang
Zhenghe
Z
Department of Genetics and Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA.
D000818:Animals; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D002874:Chromosome Mapping / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D000939:Epitopes / Q000494:pharmacology; D018390:Gene Targeting / Q000379:methods; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000235:genetics; D016678:Genome / Q000235:genetics; D006801:Humans; D015335:Molecular Probes / Q000378:metabolism / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology
D000939:Epitopes; D015335:Molecular Probes; D011506:Proteins