Tips for Successful Dual-Reporter Assays

Previously, we described some of the advantages of using dual-reporter assays (such as the Dual-Luciferase®, Dual-Glo® Luciferase and the Nano-Glo® Dual-Luciferase® Systems). Another post describes how to choose the best dual-reporter assay for your experiments. For an overview of luciferase-based reporter gene assays, see this short video:

These assays are relatively easy to understand in principle. Use a primary and secondary reporter vector transiently transfected into your favorite mammalian cell line. The primary reporter is commonly used as a marker for a gene, promoter, or response element of interest. The secondary reporter drives a steady level of expression of a different marker. We can use that second marker to normalize the changes in expression of the primary under the assumption that the secondary marker is unaffected by what is being experimentally manipulated.

While there are many advantages to dual-reporter assays, they require careful planning to avoid common pitfalls. Here’s what you can do to avoid repeating some of the common mistakes we see with new users:

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Diabetes Research: Measuring the Activity of Insulin

We rely on insulin supplied by our pancreas at the right dose and at the right time to control our blood glucose levels and energy storage. Insulin works by regulating the energy usage of various cell types in the body. When this process goes awry, it can cause diabetes.

There are two types of diabetes, defined by how insulin is dysregulated. In Type 1 Diabetes (T1D), the pancreas produces too little insulin. Patients need to give themselves insulin in order to respond to glucose in the diet. In Type 2 Diabetes (T2D), patients do not respond well to the insulin produced in their body. Therefore, they need to give themselves more to avoid hyperglycemia (high blood glucose).

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NanoLuc: Tiny Tag with a Big Impact

Synthetic biology—genetically engineering an organism to do or make something useful—is the central goal of the iGEM competition each year. After teams conquer the challenge of cloning their gene, the next hurdle is demonstrating that the engineered gene is expressing the desired protein (and possibly quantifying the level of expression), which they may do using a reporter gene.

Reporters can also play a more significant role in iGEM projects when teams design their organism with reporter genes to detect and signal the presence of specific molecules, like environmental toxins or biomarkers. Three of the iGEM teams Promega sponsored this year opted to incorporate some version of NanoLuc® Luciferase into their projects.

NanoLuc® luciferase is a small monomeric enzyme (19.1kDa, 171 amino acids) based on the luciferase from the deep sea shrimp Oplophorus gracilirostris. This engineered enzyme uses a novel substrate, furimazine, to produce high-intensity, glow-type luminescence in an ATP-independent reaction. Unlike other molecules for tagging and detecting proteins, NanoLuc® luciferase is less likely to interfere with enzyme activity and affect protein production due to its small size.

NanoLuc® Luciferase has also been engineered into a structural complementation reporter system, NanoBiT® Luciferase, that contains a Large subunit (LgBiT) and two small subunit options: low affinity SmBiT and high affinity HiBiT. Together, these NanoLuc® technologies provide a bioluminescent toolbox that was used by the iGEM teams to address a diverse set of biological challenges.

Here is an overview of each team’s project and how they incorporated NanoLuc® technology.

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Promega Scientists Helping Researchers and Students at the Marine Biological Laboratory

This summer, I had the opportunity to go to the Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts. MBL was founded in 1888 as an institution that focuses on research and education. Woods Hole is located on Cape Cod and has rich biodiversity that is the focus of the resident researchers and the many others that travel there each summer. It was here that new model organisms were discovered, allowing significant advancement in various fields. For example, squid have large axons that allowed researchers to expand our knowledge of neurons.

Over 500 scientists from over 300 institutions in over 30 countries come to MBL each year as trainees1. There are 19 advanced research training courses for pre-and post-doctoral scientists in development, reproduction, cell physiology, microbiology, infectious disease, neuroscience, and microscopy. Faculty that teach the courses are leaders in their respective fields. In addition, MBL has a neuro-physiology fellowship program through the Grass Foundation that allows early-stage researchers to come to MBL for 14 weeks to do research.

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Go with Your Gut: Understanding How the Microbiome and Diet Influence Health

microbiome_mouse_model

Over the past decade, microbiome research has provided key insights into the relationship between our gut and our health. There are trillions of organisms in our gut, comprising the microbiome that complements our human biology, distinct from our genome. These gut microbes affect us in many ways, from affecting our mental health to our ability to fight cancer.

At the University of Wisconsin-Madison, Federico Rey and his research group are trying to understand how our diet might help or harm the important microbial communities in our gut. “If we can understand how microbes interact with diet, we can personalize nutrition to match diet with the composition of the gut microbiome and promote health,” Rey says.

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Out-FOXOing High-Stage Neuroblastoma

Fluorescence microscopy of neuroblastoma cells.

In recent years, scientists have been hot on the trail of transcription factor FOXO3, tracing its involvement in various tumor-centric activities comprising the many trademarks of cancer, from drug resistance to metastasis to tumor angiogenesis.

FOXO3 is a member of the O sub-class of the forkhead box family of transcription factors. The forkhead box (FOX) family is characterized by a fork head DNA-binding domain (DBD), comprised of around 100 amino acids. They have also proven themselves to be a family of many hats, functioning in diverse roles ranging from metabolism, immunology, cell-cycle control, development, as well as cancer (1). The forkhead box O (FOXO) sub-class alone has demonstrated involvement in a variety of cellular outcomes, from drug resistance and longevity to apoptosis induction.

Due to its pro-apoptotic and anti-proliferative proclivity, FOXO3 has been previously identified as a tumor suppressor gene. However, more and more studies have begun to flip the narrative on FOXO3, portraying it more as a devoted henchman, due to its roles in drug and radiotherapy resistance, cell-cycle arrest and long-term maintenance of leukemia-initiating stem cells in a variety of cancer types, including breast cancer, pancreatic cancer, glioblastoma, and both acute and chronic myeloid leukemia.

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Targeted Gene Modification in Prairie Voles Using CRISPR and pGEM®-T Easy Vectors

As the number of children diagnosed with autism spectrum disorder (ASD) continues to rise, the search for a cause continues. Scientists have been studying genetically modified oxytocin receptors, which have shown promise as a target for studying ASD-related behaviors. One of the obstacles to designing robust scientific experiments for investigating potential ASD causes or treatments is the lack of a truly appropriate model organism for social behaviors in humans (1). Sure, there are the traditional lab rats and lab mice that demonstrate a certain level of social behaviors. However, there has been a loss of natural social behaviors in common lab mice strains because of the reduction in genetic complexity from inbreeding and adaptation to captivity (2). These animals cannot fully represent the depth of human social behaviors, including the ability of humans to form lasting social bonds (1).

Enter: The prairie vole (Microtus ochrogaster).

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Reliable DNA Purification from 3D Cell Cultures

Traditionally, scientists have relied on flat, two-dimensional cell cultures grown on substrates such as tissue culture polystyrene (TCPS) to study cellular physiology. These models are simple and cost-effective to culture and process. Within the last decade, however, three-dimensional (3D) cell cultures have become increasingly popular because they are more physiologically relevant and better represent in vivo conditions.

A spheroid of ~1,000 human liver cells. Image provided by Insphero.
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Choosing a Tag for Your Protein

Flow diagram for purifying HaloTag fusion proteins
Overview of the HaloTag® Mammalian Protein Purification System.

You have identified and cloned your protein of interest, but you want to explore its function. A protein fusion tag might help with your investigation. However, choosing a tag for your protein depends on what experiments you are planning. Do you want to purify the protein? Would you like to identify interacting proteins by performing pull-down assays? Are you interested in examining the endogenous biology of the protein? Here we cover the advantages and disadvantages of some protein tags to help you select the one that best suits your needs.

Affinity Tags

The most commonly used protein tags fall under the category of affinity tags. This means that the tag binds to another molecule or metal ion, making it easy to purify or pull down your protein of interest. In all cases, the tag will be fused to your protein of interest at either the amino (N) or carboxy (C) terminus by cloning into an expression vector. This protein fusion can then be expressed in cells or cell-free systems, depending on the promoter the vector contains. Continue reading “Choosing a Tag for Your Protein”

To Sleep, Perchance to Clean

While you and I are getting some shut eye each night, things are happening in our brains. Good things. Therapeutic things.

Think of it as brainwashing of a sort. There is a multiplicity of brain activities going on during sleep, and a November 1 paper in Science shows for the first time when and where in the brain these activities occur, and how they are connected.

CSF washes through the brain.

Here’s a bit of backstory.

To assess both the progression and pathogenesis of Alzheimer’s disease (AD), as well as the efficacy of AD drugs in clinical trials, there has been interest in the concentrations of amyloid-beta (Aβ) and tau protein in cerebral spinal fluid (CSF).

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