title: Regulatable in vivo biotinylation expression system in mouse embryonic stem cells.
abstract: Embryonic stem (ES) cells have several unique attributes, the two most important of which are they can differentiate into all cell types in the body and they can proliferate indefinitely. To study the regulation of these phenomena, we developed a regulatable in vivo biotinylation expression system in mouse ES cells. The E. coli biotin ligase gene BirA, whose protein product can biotinylate a 15-aa peptide sequence, called the AviTag, was cloned downstream of an IRES. The primary vector containing the doxycycline controlled transactivator gene tTA and IRES-BirA was knocked into the ROSA26 locus by homologous recombination. The secondary vector containing the AviTag tagged hKlf4 gene was exchanged into the ROSA26 locus using Cre recombinase. Western blot analysis showed that the doxycycline induced BirA protein can biotinylate the doxycycline induced AviTag tagged hKlf4 protein. The induction of hKlf4 repressed cell growth in the presence or absence of LIF. Chromatin immunoprecipitation assays using streptavidin beads showed that the AviTag tagged hKlf4 protein could enrich the Nanog enhancer. Our results suggested that the regulatable biotinylation system is promising for the gene function studies in mouse ES cells.
authors.affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Qin
authors.initials: Q
authors.lastname: Wang
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Wagner
authors.forename: Austin J
authors.initials: AJ
authors.lastname: Cooney
mesh_terms: D000818:Animals; D019921:Biotinylation / Q000187:drug effects / Q000235:genetics*; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D047369:Chromatin Immunoprecipitation; D004318:Doxycycline / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D055879:Gene Knock-In Techniques; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism; D051379:Mice; D011506:Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D029968:Escherichia coli Proteins; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D011506:Proteins; C472408:ROSA22 protein, mouse; D012097:Repressor Proteins; D019809:Streptavidin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli; D013752:Tetracycline; D004318:Doxycycline