reporter assay

Nano, Nano: Tiny Lipid Particles with Big Therapeutic Potential

Posted on by Ken Doyle
cell-transfection-viafect-luciferase-assay

Getting DNA or RNA into cells can be a tricky business, and a variety of transfection reagents have been developed over the years to make the process easier. Lipid-based reagents are especially popular because they combine efficient transfection with relatively low toxicity.

When it comes to transfection, it pays to think small. Human cells range in volume from 20–40 µm3 (sperm cells) to as large as 4 million µm3 (mature egg cells, or oocytes). For several decades, transfection reagents have targeted this size range. However, breakthrough research involves leaving the “micro” realm and entering a world that was once the domain only of science fiction: nanotechnology.

Continue reading “Nano, Nano: Tiny Lipid Particles with Big Therapeutic Potential”

From Drug Screening to Agriculture to Cardiac Development, A Dual-Luciferase Reporter Brings You the Story

Posted on by Promega

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.

Continue reading “From Drug Screening to Agriculture to Cardiac Development, A Dual-Luciferase Reporter Brings You the Story”

Shining Light on a Superbug: Clostridium difficile

Posted on by Darcia Schweitzer

Antibiotic-resistant bacteria and their potential to cause epidemics with no viable treatment options have been in the news a lot. These “superbugs,” which have acquired genes giving them resistance to common and so-called “last resort” antibiotics, are a huge concern as effective treatment options dwindle. Less attention has been given to an infection that is not just impervious to antibiotics, but is actually enabled by them.

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Clostridium difficile Infection (CDI) is one of the most common healthcare-associated infections and a significant global healthcare problem. Clostridium difficile (C. diff), a Gram-positive anaerobic bacterium, is the source of the infection. C. diff spores are very resilient to environmental stressors, such as pH, temperature and even antibiotics, and can be found pretty much everywhere around us, including on most of the food we eat. Ingesting the spores does not usually lead to infection inside the body without also being exposed to antibiotics.

Individuals taking antibiotics are 7-10 times more likely to acquire a CDI. Antibiotics disrupt the normal flora of the intestine, allowing C. diff to compete for resources and flourish. Once exposed to the anaerobic conditions of the human gut, these spores germinate into active cells that embed into the tissue lining the colon. The bacteria are then able to produce the toxins that can cause disease and result in severe damage, or even death.

Continue reading “Shining Light on a Superbug: Clostridium difficile”

A NanoBRET™ Biosensor for GPCR:G protein Interaction with the Kinetics and Temporal Resolution of Patch Clamping

Posted on by Michele Arduengo, PhD

Electrophysiologists are talented scientists/artists who see into the events of the cell with amazing detail.
Electrophysiology experiments provide a view into the cell with amazing detail. The paper reviewed here describes a molecular reporter biosensor (NanoBRET) that can offer the same kind of temporal and spatial resolution traditionally reserved for extremely labor-intensive experiments like patch clamp analysis.

I confess that I struggled through biophysics, and my Bertil Hille textbook Ion Channels of Excitable Membranes lies neglected somewhere in a box in my basement (I have not tossed it into the recycle bin—I can’t bear too, I spent too much time bonding with that book in graduate school).

My struggles in that graduate class and my attendance at the seminars of my grad school colleagues who were conducting electrophysiological studies left me with a sincere awe and appreciation of both the genius and the artistry required to produce nice electrophysiology data. The people who are good at these experiments are artists—they have the golden touch when it comes to generating that megaohm seal between a piece of cell membrane and a finely pulled glass pipette. And, they are brilliant scientists, they really understand the physics, the chemistry and the biology of the cells they study from a perspective that very few scientists ever develop.

Electrophysiology data, which often demonstrate the gating of a single channel protein in response to a single stimulus in real time–ions crossing a membrane through a single protein–are amazing for their ability, unlike virtually any other experimental data for the story they can tell about what is going on in a cell in real time under physiological conditions.

So when I read the paper recently published by Mashuo et al. in Science Signaling “Distinct profiles of functional discrimination among G proteins determine the action of G protein-coupled receptors”, this sentence really caught my attention:

When constructs were ectopically expressed in HEK 293T/17 cells, we obtained very similar kinetics for the GPCR-driven responses between NanoBRET™ biosensors and the patch clamp recordings.

They continue:

Indeed, the activation rates that we observed were very similar to those of GPCR-stimulated GIRKs [G protein-coupled, inwardly rectifying K+ channel] in native cells, suggesting that the conditions of this assay closely match the in vivo setting. This finding further demonstrates the ability of the system to resolve the fast, physiological relevant kinetics of GPCR signaling.

A reporter biosensor that can resolve events similarly to patch clamping?!  Amazing. Continue reading “A NanoBRET™ Biosensor for GPCR:G protein Interaction with the Kinetics and Temporal Resolution of Patch Clamping”

Choosing Primary and Control Reporters for Dual-Luciferase Assays

Posted on by Isobel Maciver

Dual-Reporter Assays give scientists the ability to simultaneously measure two reporter enzymes within a single sample. In dual assays, the activity of an experimental reporter is correlated with the effect of specific experimental conditions, while the activity of a control reporter relays the baseline response, providing an essential internal control that reduces variability caused by differences in cell viability or transfection efficiency. The Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay provides a choice of two sensitive reporters (firefly and NanoLuc luciferases) for use in dual-assay format. Both reporters give state-of-the-art functionality, raising the question “Which luciferase should be the primary reporter and which should be the control?”

This infographic outlines the various NanoDLR dual-reporter assay choices and the situations where you would choose one format over another. Continue reading “Choosing Primary and Control Reporters for Dual-Luciferase Assays”

Choosing the Best Luciferase Vector for Your Experiment—Now Made Easier with the Vector Selector

Posted on by Michele Arduengo, PhD

4621CAGenetic reporters are used as indicators to study gene expression and cellular events coupled to gene expression. They are widely used in pharmaceutical and biomedical research and also in molecular biology and biochemistry. Typically, a reporter gene is cloned with a DNA sequence of interest into an expression vector that is then transferred into cells. Following transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. A good reporter gene can be identified easily and measured quantitatively when it is expressed (in the organism or cells of interest).

Bioluminescent reporters are ideal for these types of studies because they have a number of important features including:
• Measurements that are almost instantaneous
• Exceptional sensitivity
• A wide dynamic range
• Typically no endogenous activity in host cells to interfere with quantitation

However, one factor that is critical for the success of a bioluminescent reporter assay is the vector.

At Promega we offer several different luciferases as reporters, and the genes for those luciferases are available in a variety of vectors. The vectors may vary in the promoters used or the presence or absence of sequences for rapid degradation. Often seemingly small changes in the vector can make a big difference in the suitability of the vector for a given experimental system. Do you need a reporter with a short half-life to detect rapid changes in gene expression? Are you studying a specifically localized protein? Do you wish to perform a transient or stable transfection?

To make finding the best reporter vector for your experimental system easy, we have developed the Luciferase Reporter Vector Selector. Using this online tool, you can narrow the choices of available vectors by promoter type, application (in vivo imaging, cancer pathway analysis, etc), availability of selectable marker, and type of luciferase.

So, as you design your luciferase reporter experiment, keep in mind this handy tool to help you choose the best luciferase vector for your needs.

Genome Editing and Reporter Technologies Enable Endogenous Pathway High-Throughput Assays

Posted on by Promega

ImageSource=RCSB PDB; StructureID=1qpf; DOI=http://dx.doi.org/10.2210/pdb1qpf/pdb;
ImageSource=RCSB PDB; StructureID=1qpf; DOI=http://dx.doi.org/10.2210/pdb1qpf/pdb;


This article review was written by guest author, Amy Landreman, in the Cellular Analysis and Proteomics Group at Promega.

Charcot-Marie Tooth (CMT) disease is one of the most common inherited neurological disorders affecting approximately 2.8 million people worldwide. The most common form of CMT, CMT Type 1A, is caused by a 1.5Mb genomic duplication on Chr17 that results in trisomy of the critical myelin gene Peripheral Myelin Protein 22 (Pmp22). The extra copy of Pmp22 results in excessive PMP22 protein causing the neurophathy associated with CMT type 1A. Although there is no way to remove the extra copy of the gene, even subtle decreases in Pmp22 expression have shown promise against this inherited neuropathy in laboratory models.

In a recent paper, Inglese et al. 2014, describe an interesting new approach used to identify compounds that effectively decrease Pmp22 expression using a novel gene editing strategy and reporter-based screen. Their challenge was to create an assay that accurately represented endogenous Pmp22 expression including both transcriptional and post-transcriptional regulatory mechanisms, while maintaining the sensitivity required to detect subtle changes in expression in a loss of signal assay in a format compatible with microtiter 1536-well quantitative high-throughput screening (qHTS). Continue reading “Genome Editing and Reporter Technologies Enable Endogenous Pathway High-Throughput Assays”

Using NanoLuc® Luciferase to Study Interactions between Environmental Flavobacteria and Mosquitos

Posted on by Michele Arduengo, PhD
nanoluc

Studies of the larval stages of Aedes triseriatus  (Eastern TreeHole Mosquito) indicate that the “tree hole” habitats in which these larva develop contain diverse microflora including the flavobacteria Elizabethkingia and Chryseobacterium. Extracts from these bacteria have many properties that might affect mosquito health, including antibacterial and anti-fungal activities. Understanding how these bacteria affect larval mosquito development has the potential to inform strategies for mosquito control.

Some initial work has been done by expressing Bacillus larvacidal toxins in some species of Gram-negative bacteria. However, only limited success was achieved using laboratory bacterial strains for such studies. Using environmental flavobacteria might prove to be a more useful approach. However, few molecular tools exist to study environmental flavobacteria. GFP reporters have been used to look at larval feeding, but autofluorescence in the pupae limit the usefulness of GFP-labeled strains for quantitative studies. Furthermore, environmental flavobacteria have unique transcription and translation machinery, and selectable markers and expression plasmids from proteobacteria do not function in these wild strains.

Chen and colleagues set out to generate molecular tools to study Flavobacterium hibernum, a fast-growing bacterium from native mosquito habitats. Their goal was to use these tools to see if A. triseratus larvae ingest and digest these bacteria and to test whether or not F. hibernum can be used to as a vector for larvacidal toxins directed against mosquito larvae. The results of their work were published in Applied and Environmental  Microbiology .

To develop a reporter that avoided issues of autofluorescence background for quantitative studies on the feeding behavior, the researchers turned to NanoLuc® Luciferase, a small, bright luciferase derived from the sea shrimp Oplopphorus gracilirostris. This luciferase has been used in mammalian cells for many kinds of studies, but it has not been used as a reporter in bacterial cells prior to the work of Chen et al. They also looked at work with laboratory flavobacteria strains that used a promoter of outer membrane protein A (PompA) to drive reporter expression as a potential system that might also work with environmental flavobacteria strains.

Continue reading “Using NanoLuc® Luciferase to Study Interactions between Environmental Flavobacteria and Mosquitos”

ADCC Reporter Bioassay Makes Top 10 Innovator List

Posted on by Michele Arduengo, PhD
The ADCC Reporter Bioassays were named a Top 10 innovation by The Scientist Magazine.
The ADCC Reporter Bioassay systems were named a Top 10 innovation by The Scientist Magazine.

For the second year running a Promega technology has made The Scientist Magazine’s list of Top 10 Innovations. In 2012 NanoLuc® luciferase technology was in the spotlight; in 2013 the ADCC Reporter Bioassay took center stage.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is the main mechanism of action (MOA) of antibodies through which virus-infected or other diseased cells are targeted for destruction by components of the cell-mediated immune system. These assays are often used to assess the effectiveness of monoclonal antibody therapies during the manufacture and development of biologic drugs. The bioluminescent assays use an alternative readout at an earlier point in ADCC MOA pathway for the quantification of Fc effector function of antibody-based molecules: the activation of gene transcription through the NFAT (nuclear factor of activated T-cells) pathway in the effector cell.

The bioassay uses engineered Jurkat cells stably expressing the FcγRIIIa receptor, V158 (high affinity) variant, and an NFAT response element driving expression of firefly luciferase. The assay is MOA-based and features frozen, thaw-and-use effector cells and optimized reagents and protocol to perform a reporter-based ADCC bioassay in a single day. The bioassay correlates with classic cytotoxic ADCC assays and is a suitable replacement for these cumbersome and highly variable assays.

The novel bioassay is linear, accurate, precise and stability indicating. Moreover, the bioassay shows good linear correlation between levels of glycosylation or fucosylation and ADCC activity. All of these features indicate the assay is suitable for use across biologic drug development programs.

Piecing the Puzzle Together: Using Multiple Assays to Better Understand What Is Happening with Your Cells

Posted on by Michele Arduengo, PhD

You often need several pieces of information to really understand what is happening within a cell or population of cells. If your cells are not proliferating, are they dying? Or, are you seeing cytostasis? If they are dying, what is the mechanism? Is it apoptosis or necrosis? If you are seeing apoptosis, what is the pathway: intrinsic or extrinsic?

If you are measuring expression of a reporter gene and you see a decrease in expression, is that decrease due to transfection inefficiencies, cytotoxicity, or true down regulation of your reporter gene?

To investigate these multiple parameters, you can run assays in parallel, but that requires more sample, and sample isn’t always abundant.

Multiplexing assays allows you to obtain information about multiple parameters or events (e.g., reporter gene expression and cell viability; caspase-3 activity and cell viability) from a single sample. Multiplexing saves sample, saves time and gives you a more complete picture of the biology that is happening with your experimental sample.

What information do you need about your cells to complete the picture?
What information do you need about your cells to complete the picture?

Multiplexing assay reagents to measure biomarkers in the same sample has often been considered an application only accomplished with antibodies or dyes and sophisticated detection instrumentation. However, Promega has developed microwell plate based assays for cells in culture that allow multiplexed detection of biomarkers in the same sample well using standard multimode multiwell plate readers. Continue reading “Piecing the Puzzle Together: Using Multiple Assays to Better Understand What Is Happening with Your Cells”