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One‐step knockin for inducible expression in mouse embryonic stem cells
Author(s) -
Choi Yong Jun,
Son Mi Young,
Hasty Paul
Publication year - 2011
Publication title -
genesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.093
H-Index - 110
eISSN - 1526-968X
pISSN - 1526-954X
DOI - 10.1002/dvg.20699
Subject(s) - transgenesis , transgene , biology , transactivation , embryonic stem cell , chromatin , expression cassette , microbiology and biotechnology , gene , doxycycline , transfection , genetics , gene expression , recombinant dna , embryogenesis , vector (molecular biology) , reproductive biology , antibiotics
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. genesis 49:92–97, 2011. © 2010 Wiley‐Liss, Inc.