Optimization of regulated LTR-mediated expression

Retroviral vectors are ideally suited to the study of gene function, allowi ng efficient, stable expression. Many biological systems (e.g., cell cycle, apoptosis) require the use of regulated expression systems. We therefore d eveloped a regulated retroviral vector system, TRA99, based on a tetracycli ne transactivator-dependent LTR, where the MMLV enhancer was replaced with a tetracycline-response element. Using fluorescence-activated flow cytometr ic analysis of a destabilized green fluorescent protein to monitor expressi on levels, we optimized the minimal promoter configuration with respect to both activated and repressed transcription, The TRA99 vectors demonstrate r egulated expression with activated levels comparable to those of standard r etroviral vectors and repressed levels indistinguishable from background. T his was achieved without using an internal promoter cassette, thus retainin g the cis-packaging elements requisite for helper-mediated transfer. (C) 20 00 Academic Press.