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Gene. 2004 Feb 18;327(1):61-73.

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.

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  • 1Biochemistry and Molecular Biology Department, Drug Discovery Division, Southern Research Institute, 2000 Ninth Ave South, Birmingham, AL 35255, USA.

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 rtTA variant 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 beta-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.

PMID:
14960361
[PubMed - indexed for MEDLINE]
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