In vitro screen to identify silent but activatable (S/A) integration sites for a tetracycline-inducible transgene in mice

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 entry site (IRES) sequence, which allows the initiation of translation in a cap-independent manner.