Reporter Gene Assay
The promoter region of a gene is the crucial regulatory cis-element which controls the transcription of the gene. The purpose of the reporter gene assay is to measure the regulatory potential of an unknown DNA-sequence (usually a promoter) by linking the sequence to an easily detectable reporter gene. Common reporter genes are GFP, β-galactosidase, β-glucuronidase and luciferase. Various detection methods are used to measure expressed reporter gene protein. These include luminescence, absorbance and fluorescence.
Principle
First, the DNA sequence of interest (ex. promoter) is cloned upstream of a reporter gene (ex. Luciferase). The recombinant reporter plasmid often contains a selectable marker, such as antibiotic resistance, to ensure a pure population after the plasmid is transfected into a cell line. Following successful transfection, expression of the reporter gene is measured.
Use #1: Gene expression Assay
Reporter genes can also be used to assay for the expression of the gene of interest. The reporter is directly attached to the gene of interest to create a gene fusion. The two genes are under the same promoter elements and are transcribed into a single messenger RNA molecule. The mRNA is then translated into protein. In these cases it is important that both proteins be able to properly fold into their active conformations and interact with their substrates despite being fused. In building the DNA construct, a segment of DNA coding for a flexible polypeptide linker region is usually included so that the reporter and the gene product will only minimally interfere with one another.
Use #2: Promoter Assay
Reporter genes can be used to assay for the activity of a particular promoter in a cell or organism. In this case there is no separate "gene of interest"; the reporter gene is simply placed under the control of the target promoter and the reporter gene product's activity is quantitatively measured. The results are normally reported relative to the activity under a "consensus" promoter known to induce strong gene expression.
Example of Application in RB field: RB-dependent repression of target promoter
pRB is able to repress the p107 promoter when overexpressed. These effects are mediated by E2F binding sites within the p107 promoter as pRB does not have DNA binding activity and must rely on E2Fs to localize to target gene promoters. This is illustrated in RB-negative primary cells which demonstrate increased p107 expression.
To assess repressive activities of pRB mutants, p107-Luciferase expression vectors are transfected into RB-negative cells (ex. Saos-2 cells), followed by transfection of an expression vector encoding pRB or the pRB mutant. By measuring expression of the reporter (Luciferase) one can determine how effective pRB or the pRB mutant is at repressing the p107 promoter.
Example of Application in RB field: RB-dependent block of E2F-driven activation of target promoter
Over-expressed pRB is able to repress E2F-driven activation of target genes. Promoters of E2F target genes often contain E2F-binding sites which serve as enhancer elements as they recruit the E2F transcription factors, which subsequently recruit subunits of the RNA polymerase II holoenzyme.
To assess repressive activities of pRB mutants over E2F-driven activation, E2F4B-Luciferase expression vectors are transfected into RB-negative cells (ex. Saos-2 cells), followed by transfection of expression vectors encoding DP and E2F, and finally pRB or the pRB mutant. The promoter of the E2F4B-Luciferase construct contains four E2F binding sites and exhibits elevated luciferase activity when transfected with E2F/DP1, corresponding to E2F-driven activation of the reporter gene. By measuring expression of the reporter (Luciferase) after transfection of the pRB expression vector, one can determine how effective pRB or the pRB mutant is at repressing the E2F-driven activation of the target promoter.
Principle
First, the DNA sequence of interest (ex. promoter) is cloned upstream of a reporter gene (ex. Luciferase). The recombinant reporter plasmid often contains a selectable marker, such as antibiotic resistance, to ensure a pure population after the plasmid is transfected into a cell line. Following successful transfection, expression of the reporter gene is measured.
Use #1: Gene expression Assay
Reporter genes can also be used to assay for the expression of the gene of interest. The reporter is directly attached to the gene of interest to create a gene fusion. The two genes are under the same promoter elements and are transcribed into a single messenger RNA molecule. The mRNA is then translated into protein. In these cases it is important that both proteins be able to properly fold into their active conformations and interact with their substrates despite being fused. In building the DNA construct, a segment of DNA coding for a flexible polypeptide linker region is usually included so that the reporter and the gene product will only minimally interfere with one another.
Use #2: Promoter Assay
Reporter genes can be used to assay for the activity of a particular promoter in a cell or organism. In this case there is no separate "gene of interest"; the reporter gene is simply placed under the control of the target promoter and the reporter gene product's activity is quantitatively measured. The results are normally reported relative to the activity under a "consensus" promoter known to induce strong gene expression.
Example of Application in RB field: RB-dependent repression of target promoter
pRB is able to repress the p107 promoter when overexpressed. These effects are mediated by E2F binding sites within the p107 promoter as pRB does not have DNA binding activity and must rely on E2Fs to localize to target gene promoters. This is illustrated in RB-negative primary cells which demonstrate increased p107 expression.
To assess repressive activities of pRB mutants, p107-Luciferase expression vectors are transfected into RB-negative cells (ex. Saos-2 cells), followed by transfection of an expression vector encoding pRB or the pRB mutant. By measuring expression of the reporter (Luciferase) one can determine how effective pRB or the pRB mutant is at repressing the p107 promoter.
Example of Application in RB field: RB-dependent block of E2F-driven activation of target promoter
Over-expressed pRB is able to repress E2F-driven activation of target genes. Promoters of E2F target genes often contain E2F-binding sites which serve as enhancer elements as they recruit the E2F transcription factors, which subsequently recruit subunits of the RNA polymerase II holoenzyme.
To assess repressive activities of pRB mutants over E2F-driven activation, E2F4B-Luciferase expression vectors are transfected into RB-negative cells (ex. Saos-2 cells), followed by transfection of expression vectors encoding DP and E2F, and finally pRB or the pRB mutant. The promoter of the E2F4B-Luciferase construct contains four E2F binding sites and exhibits elevated luciferase activity when transfected with E2F/DP1, corresponding to E2F-driven activation of the reporter gene. By measuring expression of the reporter (Luciferase) after transfection of the pRB expression vector, one can determine how effective pRB or the pRB mutant is at repressing the E2F-driven activation of the target promoter.