In Vitro Screen to Identify Silent but Activatable (S/A) Integration Sites for a Tetracycline-Inducible Transgene in Mice

Cold Spring Harbor Protocols
Richard BehringerAndras Nagy


To obtain ubiquitous or simply widespread transgene expression from a single stable integrant transgene is quite challenging because the random genomic integration sites of transgenes may create expression variation or frequent silencing. The tetracycline (Tet)-inducible system requires two reliable working transgenes, one for the tetracycline transactivators (tTA or rtTA) and one for the responder transgene driven by the tet-O promoter. Therefore, the challenge of getting this system working properly is a serious prospect. In this protocol, we describe how to identify a silent but highly activatable genomic site by taking advantage of transgenic lines reliably expressing the tetracycline transactivators from the Rosa-26 locus. These lines provide optimal Tet-inducible expression: There is minimal leakiness at the "off" state and a high level of induction in the presence of the inducer, doxycycline. The procedure requires (1) an embryonic stem (ES) cell line (germline competent) expressing rtTA from the Rosa-26 locus and (2) construction of a Tet-inducible transgene. The transgene contains the tet-O promoter followed by the gene of interest linked to a βgeo gene (a fusion between lacZ and neo) through an internal ribosomal entr...Continue Reading


Jan 1, 2007·CSH Protocols·Andras NagyRichard Behringer
Jan 1, 2006·CSH Protocols·Andras NagyRichard Behringer
Jan 1, 2006·CSH Protocols·Andras NagyRichard Behringer
Jan 1, 2006·CSH Protocols·Andras NagyRichard Behringer
Jan 1, 2006·CSH Protocols·Andras NagyRichard Behringer

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