Studying Gene Regulation with the LightSwitch System

Use SwitchGear 3’UTR GoClones for:

Quantifying the impact of the 3’UTR in post-transcriptional gene regulation
Measuring the effect of a miRNA or siRNA on the regulation of transcript stability or translation efficiency
Measuring the effect of sequence variants on 3’UTR function (see our Sequence Variant Assay Services)

Use SwitchGear Promoter GoClones for:

Understanding the mechanism by which transcription is induced or repressed
Characterizing the functional consequences of transcription factor binding
Screening full regulatory networks with SwitchGear’s Pathway Screening Panels
Measuring the effect of sequence variants on promoter function (see our Sequence Variant Assay Services)

ADVANTAGES:

Optimal performance: Novel RenSP luciferase technology allows you to measure promoter or 3’UTR activity with industry-leading sensitivity and dynamic range
Simple, quick, complete solution: Complete LightSwitch Luciferase Assay System enables easy transfection of luciferase reporter constructs and controls followed by detecting reporter signal with optimized LightSwitch Luciferase Assay Reagents
Comprehensive and verified: Choose from a human genome-wide collection of 30,000 transfection-ready sequence-verified promoter and 3'UTR reporter constructs. No cloning, DNA preparation, or reagent optimization is needed

OVERVIEW

Control of gene expression in eukaryotic cells is regulated on transcriptional and post-transcriptional levels. Transcription factors are important regulators of transcription rates, and microRNAs are key mediators of mRNA stability and translation efficiency.

Regulation of transcription

DNA-encoded elements like promoters interact with transcription factors and other regulatory proteins to determine when, where, and how much of a gene’s mRNA product gets made. Reporter assays are a powerful technique for measuring the activity of promoters in living cells. Promoter GoClone Reporter Constructs are created by isolating a promoter from the human genome and cloning it upstream of the RenSP luciferase reporter gene on a plasmid. Once this plasmid is transfected into a living cell, a change in promoter activity causes a change in reporter signal (light output).

SwitchGear Promoter GoClone Reporter Assays can be used to monitor the activity of any promoter in a variety of conditions to understand how its activity is regulated. Researchers may choose to examine promoter activity upon over-expressing or knocking down the activity of a transcription factor, changing the cell’s surroundings by adding a compound or changing its environment, inducing differentiation, or any number of additional condition changes. Promoter GoClone Reporter Assays are also a useful tool for studying how promoter activity changes in the presence of naturally occurring sequence variants or after site-directed mutagenesis of a motif of interest.

Regulation of mRNA stability and translation efficiency

A gene’s total protein output may also be regulated by changing the stability of an mRNA transcript or modifying the efficiency with which that transcript is translated. Small RNAs are important mediators of post-transcriptional regulation and include endogenous or exogenous microRNAs (miRNAs) and short interfering RNAs (siRNAs). These small RNAs often bind to complementary sequences in the 3’UTRs of target mRNAs resulting in increased mRNA degradation or repression of translation efficiency.

Reporter assays are a powerful technique for measuring the activity of 3’UTRs in living cells. 3’UTR GoClone Reporter Constructs are created by isolating a 3’UTR from the human genome and cloning it downstream of the constitutively expressed RenSP luciferase reporter gene on a plasmid. Once this plasmid is transfected into a living cell, a change in regulation at the 3’UTR causes a change in reporter signal (light output).

SwitchGear 3’UTR GoClone Reporter Assays can be used to assess the impact of changes in cellular conditions, including changes in miRNA or siRNA activity, on post-transcriptional regulation. While technologies like qPCR detect effects on mRNA stability, the output from a LightSwitch Reporter Assay reflects changes in both mRNA stability and translation efficiency. 3’UTR GoClone Reporter Assays are also a unique tool for measuring the impact of sequence variants on 3’UTR function.

RESOURCES
Protocols
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