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Bioluminescence Monitoring of Promoter Activity In Vitro and In Vivo

Delhove, JMKM; Karda, R; Hawkins, KE; FitzPatrick, LM; Waddington, SN; McKay, TR; (2017) Bioluminescence Monitoring of Promoter Activity In Vitro and In Vivo. In: Gould, D, (ed.) Mammalian Synthetic Promoters. (pp. 49-64). Humana Press: New York, USA.

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Abstract

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.

Type: Book chapter
Title: Bioluminescence Monitoring of Promoter Activity In Vitro and In Vivo
Location: United States
ISBN: 978-1-4939-7221-0
DOI: 10.1007/978-1-4939-7223-4_5
Publisher version: http://doi.org/10.1007/978-1-4939-7223-4_5
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: Bioluminescence, Lentivirus, Luciferase, Promoter, Transcription factor
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Cell and Developmental Biology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Pop Health Sciences > Inst for Women's Health
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Pop Health Sciences > Inst for Women's Health > Maternal and Fetal Medicine
URI: http://discovery.ucl.ac.uk/id/eprint/1570480
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