| Autor: |
Delhove JMKM; Cardiovascular and Cell Sciences Research Institute, St. George's University of London, Cranmer Terrace, London, SW17 0RE2, UK.; Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa., Karda R; Gene Transfer Technology Group, Institute for Women's Health, University College London, 86-96 Chenies Mews, London, WC1E 6HX, UK., Hawkins KE; Cardiovascular and Cell Sciences Research Institute, St. George's University of London, Cranmer Terrace, London, SW17 0RE2, UK., FitzPatrick LM; Cardiovascular and Cell Sciences Research Institute, St. George's University of London, Cranmer Terrace, London, SW17 0RE2, UK.; School of Healthcare Science, Manchester Metropolitan University, John Dalton Building, Chester Street, Manchester, M1 5GD, UK., Waddington SN; Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.; Gene Transfer Technology Group, Institute for Women's Health, University College London, 86-96 Chenies Mews, London, WC1E 6HX, UK., McKay TR; Cardiovascular and Cell Sciences Research Institute, St. George's University of London, Cranmer Terrace, London, SW17 0RE2, UK. T.McKay@mmu.ac.uk.; School of Healthcare Science, Manchester Metropolitan University, John Dalton Building, Chester Street, Manchester, M1 5GD, UK. T.McKay@mmu.ac.uk. |
| Abstrakt: |
The application of luciferase reporter genes to provide quantitative outputs for the activation of promoters is a well-established technique in molecular biology. Luciferase catalyzes an enzymatic reaction, which in the presence of the substrate luciferin produces photons of light relative to its molar concentration. The luciferase transgene can be genetically inserted at the first intron of a target gene to act as a surrogate for the gene's endogenous expression in cells and transgenic mice. Alternatively, promoter sequences can be excised and/or amplified from genomic sources or constructed de novo and cloned upstream of luciferase in an expression cassette transfected into cells. More recently, the development of synthetic promoters where the essential components of an RNA polymerase binding site and transcriptional start site are fused with various upstream regulatory sequences are being applied to drive reporter gene expression. We have developed a high-throughput cloning strategy to develop lentiviral luciferase reporters driven by transcription factor activated synthetic promoters. Lentiviruses integrate their payload cassette into the host cell genome, thereby facilitating the study of gene expression not only in the transduced cells but also within all subsequent daughter cells. In this manuscript we describe the design, vector construction, lentiviral transduction, and luciferase quantitation of transcription factor activated reporters (TFARs) in vitro and in vivo. |
