Homogeneity and long-term stability of tetracycline-regulated gene expression with low basal activity by using the rtTA2S-M2 transactivator and insulator-flanked reporter vectors $ Zhican Qu 1 , Jaideep V. Thottassery 1 , Sabrina Van Ginkel, Marina Manuvakhova, Louise Westbrook, Carrie Roland-Lazenby, Susan Hays, Francis G. Kern * Biochemistry and Molecular Biology Department, Drug Discovery Division, Southern Research Institute, 2000 Ninth Ave South, Birmingham, AL 35255, USA Received 18 July 2003; received in revised form 9 October 2003; accepted 15 October 2003 Abstract Inducible expression of tetracycline responsive element (TRE)-regulated genes in nearly all cells in a stable clone has generally been problematic, especially in long-term culture. Heterogeneity of tet-inducible expression is generally attributed to the instability of the original tet-transactivators tTA and rtTA. These transactivators have cryptic splice sites, prokaryotic codons and full VP16 domains, all of which contribute to their instability. Moreover, they also require high concentrations of Doxycycline (Dox). The 5 amino acid substitutions in the rtTAvariant rtTA2S-M2 confer exquisite sensitivity to Dox. Moreover, humanized codons, removal of cryptic splice sites and minimal VP16 domains in rtTA2S-M2 results in its being better tolerated within cells. However, the ability of this modified transactivator to maintain homogeneous inducibility in long-term culture has not been examined. We demonstrate that rtTA2S-M2 expressing clones exhibit functional transactivator activity for over 7 months in culture. Furthermore, rtTA2S-M2 expressing clones with chromosomally integrated copies of a TRE– green fluorescent protein (GFP) reporter also exhibited homogeneous inducibility in long-term culture. Importantly, the inherent reduced toxicity and improved stability of rtTA2S-M2 obviates the need to continuously select for its message, once clones with functional transactivator are isolated. The use of rtTA2S-M2 did not, however, preclude clones with stably integrated TRE-reporter from exhibiting leakiness. However, inclusion of flanking double copies of a ‘minimal core element’ of the chicken h-globin gene insulator, instead of the 1.4 kb region, in the TRE-reporter was sufficient to markedly reduce the frequency of clones with high basal expression. Inclusion of the insulator core also did not affect the maximal expression levels of the inducible gene, which typically equaled or exceeded that observed with the strong constitutive CMV promoter. Finally, with this system homogeneous inducibility was observed rapidly and with low doses of Dox. D 2003 Elsevier B.V. All rights reserved. Keywords: Tet-on; Tet-off; Synthetic tet-transactivators; Insulator core elements 1. Introduction A system that allows the regulated expression of genes of interest in mammalian cells based on the repressor (TetR) of the Tn10 tetracycline (tet) resistance operon of Escher- ichia coli was first described by Gossen and Bujard (Gossen and Bujard, 1992). In this original system, TetR was fused to VP16, the C-terminal portion of a strong transcriptional activator from herpes simplex virus to gen- erate tTA (the Tet-off transactivator). In this system TetR binds with high affinity to tetracycline response element (TRE) (an array of operator sequences, tetO), while the fused VP16 activates transcription from a minimal CMV promoter downstream of the TRE (Gossen and Bujard, 1992). Doxycycline (Dox) prevents the interaction of TetR and TRE and can therefore be used to efficiently switch off transcription from this promoter (Gossen and Bujard, 1992). This provided a simple means of regulating expres- sion of cDNAs cloned downstream of the promoter. Sub- sequently, a TetR mutant was described which contained 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2003.10.029 Abbreviations: Dox, doxycycline; GFP, green fluorescent protein; hrGFP, humanized Renilla GFP; IRES, Internal ribosome entry site; tetR, tetracycline repressor protein; HSV VP16, herpes simplex virus virion protein 16; LacZ, h-galactosidase gene; tet, tetracycline; tTA, tet trans- activator; rtTA, reverse tet transactivator; TRE, tetracycline response element. $ The complete sequences of the three plasmids depicted in Fig. 1 are available for download online at ftp://ftp.sri.org/TET . * Corresponding author. Division of Pharmaceutical Biology, Lexicon Genetics, 8800 Technology Forest Place, The Woodlands, TX 77381, USA. Tel.: +281-863-3780. E-mail address: fkern@lexgen.com (F.G. Kern). 1 These authors contributed equally to the work. www.elsevier.com/locate/gene Gene 327 (2004) 61 – 73