Today’s blog was written by guest blogger Katarzyna Dubiel, marketing intern in Cellular Analysis and Proteomics. Last updated 02/12/2021
Reporter gene assays have been critical for the study of a wide-range of biological questions, from regulation of gene expression to cellular signaling. While reporter gene assays constitute a large group of technologies, here we highlight the diversity of new discoveries enabled by highly quantitative and easily measured bioluminescent luciferase-based reporter assays. Below are our top picks of exciting research discoveries involving the Dual-Luciferase Reporter Assay format using firefly and Renilla luciferases.
Drug Screening
Alzheimer disease is a neurodegenerative disease characterized by memory loss that predominantly affects the aging population. According to the National Institute on Aging, Alzheimer disease is ranked sixth as the leading cause of death in the Unites States1. The activities of two proteases ADAM10 and BACE-1 have been linked to Alzheimer disease progression. ADAM10 functions to cleave the amyloid precursor protein (APP) and generate a neuroprotective product preventing growth of amyloidogenic A-beta peptides, while BACE-1 cleavage of APP is linked to increased A-beta amyloid growth. Using the Dual-Luciferase Reporter Assay, Reinhardt et al. screened a compound library of over 600 FDA-approved drugs for increased ADAM10 activity and identified disulfiram as having both ADAM10 stimulating and BACE-1 inhibiting activity. Further, disease mouse models displayed increased ADAM10 expression, lowered levels of plaques in certain regions of the brain, and decreased behavioral symptoms upon disulfiram treatment. The time from research to clinic for disufiram may be fairly quick since it is already used to support the treatment of chronic alcoholism. Read the full article: Identification of disulfiram as a secretase-modulating compound with beneficial effects on Alzheimer’s disease hallmarks
Cancer and MicroRNAs
MicroRNAs are small (approximately 18–4 nucleotide) non-coding RNAs that function in post-transcriptional regulation of gene expression. Recently, a link between microRNAs and cancer has been identified with microRNA dysregulation leading to poorer prognosis and cancer progression. A study by Fu et al employed a large-scale microRNA search to identify 29 dysregulated miRNAs in prostate cancer. Specifically, miRNA-19a was significantly upregulated in prostate cancer patients. Fu et al. determined that increased expression of miRNA-19a stimulated cell proliferation, cell cycle progression and migration. Further, using the Dual-Luciferase Reporter Assay, they identified VPS37A (Vascular Protein Sorting 37 homolog A) as the downstream effector or miRNA-19a regulation. This research draws attention to miRNAs as potential therapeutic targets and early diagnostic biomarkers of cancer. Read the article here: MicroRNA-19a acts as a prognostic marker and promotes prostate cancer progression via inhibiting VPS37A expression
Virology
The CDC estimates at total of 30 million people in the United States fell ill with the influenza virus in the 2016-2017 flu season2. There are multiple types of influenza virus, with influenza A and B causing seasonal outbreaks each winter. Successful infrection requires specific protein-RNA interactions.. Specifically, influenza A nucleoprotein (NP) interacts with regions of the viral RNA genome and is essential for viral packaging. Using the photoactivatable ribonucleoside enhanced cross-linking and immunoprecipitation (PAR-CLIP) method, Williams et al identified the specific genomic RNA regions bound by NP. NP binds RNA non-uniformly, and regions absent of NP were predicted to form RNA secondary structures. Mutations affecting these secondary structures led to decreased infectivity and viral replication as tested by methods including the Dual-Luciferase Reporter Assay. Understanding the biological events required for infectivity can inform vaccine design, leading to more effective influenza vaccines. . Read the article here: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication
Crop Production and Yield
Current estimates show that agricultural crop production must double by 2050 to meet global demands3. To help achieve these food production goals, researchers are investigating the genetic mechanisms that control grain size. Sun et al. pinpoint a key G-protein regulated pathway involved in regulation of rice grain size and length.. Within this pathway, rice have multiple Gγ proteins that work together to determine grain size. Multiple genetic studies isolated the interplay of these proteins. GS3 is identified as a negative regulator of grain size, while DEP1 and GGC2 are positive regulators. Using multiple experimental methods, including the Dual-Luciferase Reporter Assay, the authors determined these Gγ proteins interact with RGB1 through an OSR domain to influence grain size. Importantly, similar G-protein pathways were identified in a range of plant species. Genetic manipulation of these pathways may help increase crop yields. Read the full article: A G-protein pathway determines grain size in rice
Evolution and Development
Multiple progenitor cell populations and gene expression profiles must be coordinated to form the human four-chambered heart. Mutations disrupting this process commonly lead to congenital heart defects.The research highlighted here identifies common features of the human and zebrafish heart, which were previously thought to be developmentally distinct. Guerra et al. characterized atrial-specific RNA profiles and identified multiple enriched genes including pitx2 and meis2b. Notably, Pitx2 has been associated with left atrial development in humans. Meis2b knockout zebrafish displayed irregular ratios of atrial to ventricular surface areas and cardia conduction defects. Using the Dual-Luciferase Reporter Assay, Meis2b was identified as a positive regulator working upstream of the Pitx2c protein. This work presents an evolutionary link between the two-chambered heart of bony fish and the four-chambered heart of humans and may provide insights into how congenital heart defects develop. Read the full text here: Distinct myocardial lineages break atrial symmetry during cardiogenesis in zebrafish
Since the introduction of the first bioluminescent dual-luciferase assay in 1995, this approach has been used in countless studies to advance our scientific understanding of cellular gene regulation. To learn more about the last 30 years of bioluminescent innovations and research discoveries please visit our 30th anniversary web page.
Papers Reviewed
Reinhardt, S. et al. (2018) Identification of disulfiram as a secretase-modulating compound with beneficial effects on Alzheimer’s disease hallmarks. 8,1329.
Fu, F. et al. (2018) MicroRNA-19a acts as a prognostic marker and promotes prostate cancer progression via inhibiting VBS37A expression. 9, 1931–43.
Williams, G.D. et al. (2018) Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication. 9, 465.
Sun, S. et al. (2018) A G-protein pathway determines grain size in rice. 9, 851.
Guerra, A. et al. (2018) Distinct myocardial lineages break atrial symmetry during cardiogenesis in zebrafish. May 15.
References not linked in text
- National Institute on Aging. Alzheimer’s disease fact sheet
- CDC (2018) Estimated influenza illnesses, medical visits and hospitalizations averted by vaccination in the United States
- United Nations. (2009) Food production must double by 2050 to meet demand from world’s growing population, innovated strategies needed to combat hunger, experts tell second committee
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