Doxycycline- and tetracycline-regulated transcriptional silencer enhance the expression level and transactivating performance of rtTA.

Background: The tetracycline-regulated transcriptional silencer (tTS) has been demonstrated to mitigate leaky expression of the tetracycline-inducible promoter under uninduced condition, and, when conjugated with reverse-type tetracycline-controlled transactivator (rtTA), shows great promise for gene therapy. This effect was attributed to the effectiveness of tTS as a repressor of transcription at the tetracycline-regulated promoter. However, we observed an unexpected increase in transactivational activity by rtTA in the presence of tTS under inducible condition.

Regulation of anoikis by Cdc42 and Rac1.

Ras family small GTPases play a critical role in malignant transformation, and Rho subfamily members contribute significantly to this process. Anchorage-independent growth and the ability to avoid detachment-induced apoptosis (anoikis) are hallmarks of transformed epithelial cells. In this study, we have demonstrated that constitutive activation of Cdc42 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells.

An ecdysone and tetracycline dual regulatory expression system for studies on Rac1 small GTPase-mediated signaling.

Regulated expression systems are invaluable for studying gene function, offer advantages of dosage-dependent and temporally defined gene expression, and limit possible clonal variation when toxic or pleiotropic genes are overexpressed. Previously, establishment of inducible expression systems, such as tetracycline- and ecdysone-inducible systems, required assessment of the inducible characteristics of individual clones by tedious luciferase assays. Taking advantage of a green fluorescent protein (GFP) reporter controlled by tetracycline- or ecdysone-responsive element and fluorescence-activated cell sorting, we propose a simple and efficient strategy to select highly inducible cell lines according to their fluorescence profiles after transiently transfecting the candidate cell pools with a surrogate GFP reporter.