Abstract
Promoter deletion analysis is a useful tool for identifying important regulatory regions involved in transcriptional control of gene expression. In this approach, a series of promoter deletion fragments are fused to a reporter gene, such as chloramphenicol acetyltransferase or luciferase gene in a vector, and then transfected into cells for induction. Screening the expression level of the reporter gene using either a qualitative or a quantitative assay, allows to identify the regulatory regions of interest (e.g., cis-acting elements or enhancer) in the promoter.
Luciferase genes have been widely used as reporter genes for their sensitivity and efficiency. Firefly and Renilla luciferases are two commonly used reporters, which oxidize different substrates to generate quantifiable luminescence. Therefore, the enzymatic activities of firefly and Renilla luciferases can be sequentially measured in a single sample by controlling reaction conditions. Here, we describe a dual-luciferase reporter assay, where the promoter of interest is fused to a firefly luciferase reporter and is co-transfected into cells with an internal control vector (pRL-CMV) to express Renilla luciferase. Both the Firefly and Renilla luciferases are measured using a dual-luciferase reporter assay system which improves experimental accuracy.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Riethoven JJ (2010) Regulatory regions in DNA: promoters, enhancers, silencers, and insulators. Methods Mol Biol 674:33–42
Fitzgerald PC, Shlyakhtenko A, Mir AA, Vinson C (2004) Clustering of DNA sequences in human promoters. Genome Res 14:1562–1574
Breathnach R, Chambon P (1981) Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem 50:349–383
Dynan WS, Tjian R (1985) Control of eukaryotic messenger RNA synthesis by sequence-specific DNA-binding proteins. Nature 316:774–778
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002) Molecular biology of the cell. Garland Science, New York
Bookstein R, Rio P, Madreperla SA et al (1990) Promoter deletion and loss of retinoblastoma gene expression in human prostate carcinoma. Proc Natl Acad Sci USA 87:7762–7767
Heckman CA, Cao T, Somsouk L et al (2003) Critical elements of the immunoglobulin heavy chain gene enhancers for deregulated expression of Bcl-2. Cancer Res 63:6666–6673
Soboleski MR, Oaks J, Halford WP (2005) Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells. FASEB J 19:440–442
Alam J, Cook JL (1990) Reporter genes: application to the study of mammalian gene transcription. Anal Biochem 188:245–254
Bronstein I, Fortin J, Stanley PE et al (1994) Chemiluminescent and bioluminescent reporter gene assays. Anal Biochem 219:169–181
Ow DW, de Wet JR, Helinski DR et al (1986) Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234:856–859
de Wet JR, Wood KV, Deluca M et al (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737
Fraga H, Fernandes D, Fontes R et al (2005) Coenzyme A affects firefly luciferase luminescence because it acts as a substrate and not as an allosteric effector. FEBS J 272:5206–5216
Matthews JC, Hori K, Cormier MJ (1977) Purification and properities of Renilla reniformisluciferase. Biochemistry 16:85–91
Hannah RR, Jennens-Clough ML, Wood KV (1998) Rapid luciferase reporter assay systems for high throughput studies. Promega Notes 65:9–14
Liu R, Liu H, Chen X et al (2001) Regulation of CSF1 promoter by the SWI/SNF-like BAF complex. Cell 106:309–318
Chen X, Wu J-M, Hornischer K et al (2006) TiProD: the tissue-specific promoter database. Nucleic Acids Res 34:D104–D107
Xu YZ, Thuraisingam T, Marino R, Radzioch D (2011) Recruitment of SWI/SNF complex is required for transcriptional activation of SLC11A1 gene during macrophage differentiation of HL-60 cells. J Biol Chem 286:12839–12849
Ausubel FM, Brent R, Kingston RE et al (2001) Current protocols in molecular biology, vol 1. Wiley, New York, p 2.5.3
Adkins S, Burmeister M (1996) Visualization of DNA in agarose gels as migrating colored bands: applications for preparative gels and educational demonstrations. Anal Biochem 240:17–23
Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Xu, Y.Z., Kanagaratham, C., Jancik, S., Radzioch, D. (2013). Promoter Deletion Analysis Using a Dual-Luciferase Reporter System. In: Bina, M. (eds) Gene Regulation. Methods in Molecular Biology, vol 977. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-284-1_7
Download citation
DOI: https://doi.org/10.1007/978-1-62703-284-1_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-283-4
Online ISBN: 978-1-62703-284-1
eBook Packages: Springer Protocols