Gene Promoter Reporter Vectors
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GENE PROMOTER REPORTER VECTORS
OVERVIEW
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Physiologically relevant-contains cloned promoter region of specific genes
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Flexible- Use for in vivo assays
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Quantitative- Use luciferase activity to measure promoter activity
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Safe No Radioactivity required
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Simple-Easily applied to high-throughput screening
With Panomics' Gene Promoter Reporter Vectors, you can monitor and even quantify the promoter activity of 34 genes. Each Gene Promoter Reporter Vector contains a ~1 kB insert, corresponding to the 5'-flanking sequence located approximately 800 bp upstream of exon 1 of a specific human gene and extending to the first 50-200 bp of the untranslated region in exon 1. The actual length of the cloned promoter region differs for each Gene Promoter Reporter Vector (see the table below). This insert is placed upstream of the luciferase reporter gene. Since the putative cis-acting enhancer elements are expected to exist in this cloned promoter region, the luciferase activity observed during the reporter assay more closely resembles the actual regulation of these genes within human cells.
Binding of transcription factors in this region results in the expression of firefly luciferase, an enzyme capable of catalyzing a powerful bioluminescent reaction. Light emitted from the chemical reaction is directly proportional to the amount of expressed enzyme and thus the promoter activity of each gene. The backbone of the vector contains an ampicillin resistance gene for cloning purposes, an origin of replication, and an f1 origin for single-stranded DNA production.
As a means of measuring promoter response in cells, the luciferase assay is simple, straightforward, and very effective. The reporter vector is first transfected into cells. After a limited amount of time, the cells are lysed and the substrate of luciferase, luciferin, is introduced into the cellular extract along with Mg2+ and excess ATP. Under these conditions, the luciferase enzyme expressed by the reporter vector will catalyze the oxidative carboxylation of luciferin. The luminescence from this chemical reaction can be read and quantified by a luminometer or scintillation counter. The amount of light detected from the cell lysate correlates directly with the promoter activity of each human gene
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Gene Promotor Data:HeLa cells were transfected with each of the vectors listed and then treated with/without phorbol myristate acetate (PMA). The luciferase activity showed positively regulated expression for TNFa, RB1, and JUNB Gene Promoter Vectors with PMA treatment, indicating that these three gene promoters are activated by PMA in HeLa cells while others are not.
The Gene Promoter Reporter Vector is available for 34 different genes.
The insert contains approximately 800 bp of 5' flanking region and 50-200 bp of untranslated region of exon 1 of the human genes listed below. This 1 kb region is located upstream of the start codon of luciferase gene. The vector backbone contains an ampicillin-resistance gene which allows ampicillin selection in E.coli, a pUC origin of replication for propagation in E.coli, and an f1 origin for single-stranded DNA production.
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DOCUMENTS
User Manual
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