This blog was written by Sebastien Smick, Research Technician in Dr. Jacquin Niles’ laboratory at Massachusetts Institute of Technology (MIT)
Our lab is heavily focused on the basic biology and drug discovery of the human bloodborne pathogen Plasmodium falciparum, which causes malaria. We use the CRISPR/Cas9 system, paired with a TetR protein fused to a native translational repressor alongside a Renilla luciferase reporter gene, to conditionally knock down genes of interest to create modified parasites. We can then test all kinds of compounds as potential drug scaffolds against these gene-edited parasites. Our most recent endeavor, one made possible by Promega, is a medium-low throughput robotic screening pipeline which compares conditionally-activated or-repressed parasites against our dose-response drug libraries in a 384-well format. This process has been developed over the past few years and is a major upgrade for our lab in terms of data production. Our researchers are working very hard to generate new modified parasites to test. Our robots and plate readers rarely get a day’s rest!
Bioluminescent reporter assays are an excellent choice for analyzing gene regulation because they provide higher sensitivity, wider dynamic range and better signal-to-background ratios compared to colorimetric or fluorescent assays. In a typical genetic reporter assay, cells are transfected with a vector that contains the sequence of interest cloned upstream of a reporter gene, and the reporter activity is used to determine how the target sequence influences gene expression under experimental conditions. A second control reporter encoded on the same or a different plasmid is an essential internal control. The secondary reporter is used to normalize the data and compensate for variability caused by differences in cell number, lysis efficiency, cell viability, transfection efficiency, temperature, and measurement time.
For genetic reporter assays, using a secondary control vector with a weak promoter like PGK or TK to ensures that the control does not interfere with activation of your primary reporter vector. Transfection of high amounts of the control plasmid or putting the control reporter under control of a strong promoter like CMV or SV40 often leads to transcriptional squelching or other interference with the experimental promoter (i.e., trans effects). Reporter assays can also be used to quantitatively evaluate microRNA activity by inserting miRNA target sites downstream or 3´ of the reporter gene. For example, the pmirGLO Dual-Luciferase miRNA Target Expression Vector is based on dual-luciferase technology, with firefly luciferase as the primary reporter to monitor mRNA regulation and Renilla luciferase as a control reporter for normalization.
Here in Technical Services we often talk with researchers who are just starting their project and looking for advice on designing their genetic reporter vector. They have questions like:
How much of the upstream promoter region should be included in the vector?
How many copies of a response element will be needed to provide a good response?
Does the location of the element or surrounding sequence alter gene regulation?
Each luminescent assay plate represents precious time, effort and resources. Did you know that there are three things about your detection instrument that can impact how much useful information you get from each plate? Instruments with poor sensitivity may cause you to miss low-level samples that could be the “hit” you are looking for. Instruments with a narrow detection range limit the accuracy or reproducibility you needed to repeat your work. Finally, instruments that let the signal from bright wells spill into adjacent wells allow crosstalk to occur and skew experimental results, costing you time and leading to failed or repeated experiments.
Luciferase assays are useful tools for studying a wide range of biological questions. They can be performed easily by adding a reagent that provides components necessary to generate a luminescent signal directly to cells or a cell lysate. However, once this reagent has been added, how long you wait to measure the signal becomes a key consideration in generating consistent data. Dependent on which luciferase assay you use, you may need a luminometer that can use injectors to deliver the assay reagents. The reason for this is simple, but can be confusing to new users.
Helping scientists design experiments and interpret data is what we do best at Promega Technical Services. This may mean spending time at the bench attempting to reproduce anomalous results or forming a team, perhaps with members of other departments, to brainstorm seemingly intractable experimental road blocks. Still, for many of us nothing surpasses the experience of meeting these same scientists face to face whether it be on their home turf or at a booth during a tradeshow. Continue reading “Running A Victory Lap For Promega’s Bioluminescence Technologies”
All across the plate they lay in various states of woe
And I wept there in the darkness as I watched the awful show
Apoptosis or Necrosis–’twas impossible to know
If I had a luminometer I could have made them glow. Continue reading “Lament for a Luminometer”
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