Targeting MYC: The Need to Study Protein:Protein Interactions in Cells

Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;
Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;

In 1982, picked up because of its homology to chicken virus genes that could transform cells, MYC became one of the first human genes identified that could drive cellular transformation (1,2). Since that time countless laboratories have prodded and poked the human MYC gene, the MYC protein, their homologs in other animal models, and their transforming viral counterparts.

MYC is a transcription factor and forms heterodimers with a required protein partner, MAX, before binding to the E box sequences of DNA regulatory regions (3). MYC regulates gene expression of many targets through interactions with a host of proteins, often referred to as the MYC Interactome (2).  In fact, MYC is estimated to bind 10–15% of the genome, and it regulates the expression of genes that  are transcribed by by each of the three RNA polymerases (2).

MYC plays a central role in regulating cell growth, proliferation, apoptosis, differentiation and transformation, acting as a central integrator of cellular signals. MYC is tightly regulated at multiple levels from gene expression to protein stability. Dysregulation (usually upregulation) of the amount and stability of Myc protein is observed in many human cancers. Even in cancers in which MYC is not directly involved in transforming cells, its normal expression is often required to support the extracellular matrix and/or vascularization necessary for tumor growth and formation (4).

Because MYC is such a central player cancer pathology, it is an attractive target for cancer therapeutics  (2) . Continue reading “Targeting MYC: The Need to Study Protein:Protein Interactions in Cells”

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

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.

A Little Bioluminescence for the International Year of Light

22159-2268-CR-WC-NanolucWebNavImage280x1402015 is the International Year of Light, and activities around the globe are planned to celebrate light in nature, the scientists who have helped us understand the nature of light and the engineers who have developed countless tools and technologies harnessing the power of light. At Promega, our favorite kind of light in nature is bioluminescence. So your Promega Connections bloggers thought we would share this incredible National Geographic video of ocean bioluminescence. In this video, starlight cameras capture the bioluminescence of the ocean, revealing an amazingly beautiful lightscape that is invisible to the unaided human eye. Enjoy!

Interested in Learning More? Check out these Bioluminescence-Related Blog Posts:

Angel’s Glow: Bioluminescence on the Battlefield

Bioluminescence from the Sky to the Ocean’s Depths

Tracking the Progression of the Plague Using Bioluminescence

Shedding Light on Protein:Protein Interactions with NanoBRET™ Technique

NanoBRET™ TechnologyIf you are trying to investigate protein:protein interactions inside cells, you know how important physiologically relevant results are. If you overload your cells with fusion constructs, your protein interactions may not actually reflect what is going on in the cell, and if your BRET energy donor and acceptor do not have sufficiently separated spectra, you can pick up a fair amount of noise in your experiment. Using the new superbright NanoLuc® Luciferase, and the HaloTag® Technology, we have developed a sensitive BRET system to help you take a better look specific protein interactions that interest you. Promega research scientist, Danette Daniels, describes the system in the Chalk Talk below:

Can Fruit Flies Glow in the Dark?

Fruit fly. Image from morguefile.
Question: How is a fruit fly like a firefly? No, this is not an obvious answer (their names start with the letter “f”) or the beginning of a bad entomology joke. These two organisms may both be winged insects, but as it turns out, what makes the firefly light show such a special treat on summer evenings is something that fruit flies, the bane of the kitchen in the summertime and annoyance for labs near Drosophila researchers, can mimic with a little help from a synthetic luciferin substrate as reported in PNAS. Continue reading “Can Fruit Flies Glow in the Dark?”

Dual-Luciferase Imaging in vivo

22159-2268-CR-WC-NanolucWebNavImage280x140We are used to seeing multicolored fluorescence images labeling either specific events or structures within cells. When compared to imaging with fluorescent methods, bioluminescence imaging methods provide the advantages of low background and subsequent higher signal to noise ratio—enhancing sensitivity. A key prerequisite for dual-imaging experiments is the ability to distinguish the signal from each event separately and clearly. However, compared to the large number of available fluorescent compounds (many spectrally distinct fluorescent proteins and dyes), there are not many different luciferases to choose from. This lack of variety has limited the capabilities of bioluminescence for imaging multiple molecular events in the same sample. Therefore, there is a need for new luciferases with substrates and emission spectra that are different from the beetle luciferases currently in widespread use.

A paper published in Molecular Imaging in October 2013 describes use of firefly and the new NanoLuc® Luciferase to image cell signaling events in cultured cells and in a mouse model system. The paper, authored by Stacer et al. of the University of Michigan, details a proof-of-concept experiment using firefly and NanoLuc luciferases to image two distinct events in the TGF-beta1 signaling pathway. Continue reading “Dual-Luciferase Imaging in vivo”

How Low Can You Go? Why the Brightness of NanoLuc® Matters

The new NanoLuc® Luciferase is a very small, very bright luciferase, making it ideal when you need a genetic reporter to act near physiological levels inside cells to reveal subtle regulatory events. In the chalk talk below, we illustrate why this is important with a p53/mdm2 example.

 

Explore more applications of NanoLuc® Luciferase

Watch this Webinar to learn even more 

 

 

The New Bright Light of Discovery

Flashing lights of luminescent creatures. You see them at dusk on hot summer nights and in pictures from the deepest, darkest parts of the oceans. For many of us they spark memories of magical summer nights. But for scientists they also have sparked ideas and discoveries. Continue reading “The New Bright Light of Discovery”

Running A Victory Lap For Promega’s Bioluminescence Technologies

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. PCArticle Continue reading “Running A Victory Lap For Promega’s Bioluminescence Technologies”

Variations on the Two-Hybrid Assay

puzzle_lightThe use of reporter genes for simple analysis of promoter activity (promoter bashing) is a well known practice. However, there are many other elegant applications of reporter technologies. One such application is illustrated in the paper by Zheng et al., published in the Sept. 2008 issue of Cancer Research. These researchers from the Hormel Institute at the University of Minnesota showed that the cyclin-dependent kinase cdk3 phosphorylates the transcription factor ATF1 and enhances its transcriptional and transactivation activity. The observed cdk/ATF1 signaling was shown to have an important role in cell proliferation and transformation. To do this they used several variations of a reporter-based two-hybrid assay. Continue reading “Variations on the Two-Hybrid Assay”