Optimization of regulated LTR-mediated expression.
Retroviral vectors are ideally suited to the study of gene function, allowing efficient, stable expression. Many biological systems (e.g., cell cycle, apoptosis) require the use of regulated expression systems. We therefore developed a regulated retroviral vector system, TRA99, based on a tetracycline transactivator-dependent LTR, where the MMLV enhancer was replaced with a tetracycline-response element. Using fluorescence-activated flow cytometric analysis of a destabilized green fluorescent protein to monitor expression levels, we optimized the minimal promoter configuration with respect to both activated and repressed transcription. The TRA99 vectors demonstrate regulated expression with activated levels comparable to those of standard retroviral vectors and repressed levels indistinguishable from background. This was achieved without using an internal promoter cassette, thus retaining the cis-packaging elements requisite for helper-mediated transfer.
Lorens
J B
JB
Rigel, Inc., 240 East Grand Avenue, South San Francisco, California 94080, USA. jblorens@rigel.com
Jang
Y
Y
Rossi
A B
AB
Payan
D G
DG
Bogenberger
J M
JM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004317:Doxorubicin / Q000494:pharmacology; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005434:Flow Cytometry; D015967:Gene Expression Regulation, Viral / Q000187:drug effects / Q000235:genetics*; D020868:Gene Silencing / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000502:physiology; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020218:Response Elements / Q000235:genetics; D020079:Terminal Repeat Sequences / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology; D014161:Transduction, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured; D019065:Virus Assembly
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004317:Doxorubicin; D013752:Tetracycline