title: One-step knockin for inducible expression in mouse embryonic stem cells.
abstract: Transgenesis enables the elucidation of gene function; however, constant transgene expression is not always desired. The tetracycline responsive system was devised to turn on and off transgene expression at will. It has two components: a doxycycline (dox)-controlled transactivator (TA) and an inducible expression cassette. Integration of these transgenes requires two transfection steps usually accomplished by sequential random integration. Unfortunately, random integration can be problematic due to chromatin position effects, integration of variable transgene units, and mutation at the integration site. Therefore, targeted transgenesis and knockin were developed to target the TA and the inducible expression cassette to a specific location, but these approaches can be costly in time, labor, and money. Here, we describe a one-step Cre-mediated knockin system in mouse embryonic stem cells that positions the TA and inducible expression cassette to a single location. Using this system, we show dox-dependent regulation of eGFP at the DNA topoisomerase 3β promoter. Because Cre-mediated recombination is used in lieu of gene targeting, this system is fast and efficient.
authors.affiliation: Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, Texas 78245-3207, USA.
authors.forename: Yong Jun
authors.initials: YJ
authors.lastname: Choi
authors.forename: Mi Young
authors.initials: MY
authors.lastname: Son
authors.forename: Paul
authors.initials: P
authors.lastname: Hasty
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004318:Doxycycline / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D055879:Gene Knock-In Techniques; D023061:Gene Order; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D058889:Molecular Typing; D011995:Recombination, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D004318:Doxycycline