Here in Technical Services we often talk with researchers at the beginning of their project about how to carefully design and get started with their experiments. It is exciting when you have selected the Luciferase Reporter Vector(s) that will best suit your needs; you are going to make luminescent cells! But, how do you pick the best way to get the vector into your cells to express the reporter? What transfection reagent/method will work best for your cell type and experiment? Do you want to do transient (short-term) transfections, or are you going to establish a stable cell line?
It’s a question I’m asked probably once a week. “What wavelength do I select on my luminometer when performing a luciferase assay?” The question is a good and not altogether unexpected one, especially for those new to bioluminescent assays. The answer is that in most cases, you don’t and in fact shouldn’t select a wavelength (the exception to this rule is if you’re measuring light emitted in two simultaneous luciferase reactions). To understand why requires a bit of an explanation of absorbance, fluorescence, and luminescence assays, and the differences among them.
Absorbance, fluorescence, and luminescence assays are all means to quantify something of interest, be that a genetic reporter, cell viability, cytotoxicity, apoptosis, or other markers. In principle, they are all similar. For example, a genetic reporter assay is an indicator of gene expression. The promoter of a gene of interest can be cloned upstream of a reporter such as β-galactosidase, GFP, or firefly luciferase. The amount of each of these reporters that is transcribed into mRNA and translated into protein by the cell is indicative of the endogenous expression of the gene of interest.
We’re all familiar with the Central Dogma of Molecular Biology: DNA is transcribed into RNA, which is translated into proteins. It’s drilled into our heads from the early days of biology classes, and it’s surprisingly useful when we start exploring in our own research projects. For example, if you’re interested in gene expression, you’ll most likely be working with RNA, specifically mRNA. Messenger RNA (mRNA) is transcribed from DNA and is used by ribosomes as a “template” for a specific protein. The total mRNA in a cell represents all of the genes that are actively being transcribed. So, if you want to know whether or not a gene is being transcribed, RNA purification is a great place to start.
When preparing your RNA samples for a downstream assay, there are several roadblocks and pitfalls that could give you quite a headache. Let’s tackle two of the most common.
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.
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”
CRISPR is a hot topic right now, and rightly so—it is revolutionizing research that relies on editing genes. But what exactly is CRISPR? How does it work? Why is everyone so interested in using it? Today’s blog is a beginner’s guide on how CRISPR works with an overview of some new applications of this technology for those familiar with CRISPR.
Introduction to CRISPR/Cas9
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were discovered in 1987, but it took 30 years before scientists identified their function. CRISPRs are a special kind of repeating DNA sequence that bacteria have as part of their “immune” system against invading nucleic acids from viruses and other bacteria. Over time, the genetic material from these invaders can be incorporated into the bacterial genome as a CRISPR and used to target specific sequences found in foreign genomes.
CRISPRs are part of a system within a bacterium that requires a nuclease (e.g. Cas9), a single guide RNA (sgRNA) and a tracrRNA. The tracrRNA recruits Cas9, while sgRNA binds to Cas9 and guides it to the corresponding DNA sequence of the invading genome. Cas9 then cuts the DNA, creating a double-stranded break that disables its function. Bacteria use a Protospacer Adjacent Motif, or PAM, sequence near the target sequence to distinguish between self and non-self and protect their own DNA.
While this system is an effective method of protection for bacteria, CRISPR/Cas9 has been manipulated in order to perform gene editing in a lab (click here for a video about CRISPR). First, the tracrRNA and sgRNA are combined into a single molecule. Then the sequence of the guide portion of this RNA is changed to match the target sequence. Using this engineered sgRNA along with Cas9 will result in a double-stranded break (DSB) in the target DNA sequence, provided the target sequence is adjacent to a compatible PAM sequence.
While scientists using ancient DNA analysis are learning how Yersinia pestis developed over time into the causative agent of three worldwide pandemics, there is still much to learn about the early hours and days of an organism infected with the plague. Y. pestis still infects humans so any insight into disease progression is useful for determining treatment timing and even developing novel treatments to supplement or replace antibiotics. A 2012 study observed how Y. pestis injected into mice spread throughout the body using bioluminescent imaging to track the infection. More recent research reported in PLOS ONE used a more real-world route of infection by introducing an aerosolized Y. pestis to a nonhuman primate model and tracking the transcripts altered during the first 42 hours of infection. Continue reading “Analyzing the Effects of Yersinia pestis Infection on Gene Expression”
The ideal cancer detection method would involve giving a sample of blood or tissue and using DNA or RNA analysis to determine if there were any gene sequence or gene expression changes that are known hallmarks of cancer. Unfortunately, most current screening methods used are not so precise and in some cases are invasive. However useful tests for colon cancer may be, many people do not subject themselves to the standard colonoscopy. What if there was an easier, noninvasive method that could be used to screen for cancer and detect changes at the early, easily treatable stages of cancer? A recent article in PLOS ONE describes just such a mutation detection method for colorectal cancer using purified nucleic acid with a method that involves emulsion PCR, bead arrays and fluorescent probes. Continue reading “Cancer Detection on a Chip?”
Riboprobes are RNA probes that can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter.
Viruses code for their own RNA polymerases, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned gene.
Transcription of RNA is performed with the appropriate RNA polymerase (T3, T7 or SP6), depending on the RNA polymerase promoter sites present in the chosen vector. Because these polymerases are extremely promoter-specific (i.e., there is almost no transcriptional cross talk), virtually homogeneous RNA can be obtained using plasmid DNA as the template in a transcription reaction. When it is desirable to copy only insert DNA sequences, the plasmid is linearized at an appropriate restriction site before the transcription reaction and only discrete “run-off” transcripts are obtained, virtually free of vector sequences. RNA transcripts may be used to generate radioactive probes for hybridization to Northern and Southern blots, plaque and colony lifts as well as non-radioactive probes (i.e, labeled with digoxgenin)for in situ hybridization.
Protein location: outer mitochondrial membrane (Yeast in vitro import assay)
Curado, S. et.al. (2010) Dis.Mod. Mech.3, 486-95. PubMed ID 20483998.
Chemically mutagenized zebra fish were assayed for liver defects in their F3 progeny.This screen led to the identification of mutant called oliver. Oliver mutants have an o-shaped liver of a much deprived size due to the depletion of most of the hepatocytes. This mutation maps to the Tomm22gene which encodes a translocase of the outer membrane and thought to play an important role in protein import into mitochondria. Various Tomm22 mutants were expressed and used in a yeast in vitro import systemto determine if correct inserted into the yeast outer mitochondrial membrane.
Proline hydroxylation is also a vital component of hypoxia via hyposxia inducible factors. The cellular response to hypoxia involves the induction of the hypoxia-inducible factor considered to be the major transcription factor involved in gene regulation of hypoxia. This factor is hydroxylated by prolyl-hydroxase dolman proteins (PHDs). To investigate if a newly identified component of the hypoxia pathway (Elk3) is also hydroxylated, proteins were expressed +/- PHDs cofactors and protein mobility was measured via gel analysis.
Programmed -1 ribosomal frameshifting (PRF) is a distinctive mode of gene expression utilized by some viruses (HIV-1 for example). Recently a genome-wide screen demonstrated that down regulation of eukaryotic release factor (eRF1) inhibited HIV-1 replication. In order to characterize the dose dependent effect of eRF1, increasing amounts were expressed in the presence of dual luciferase reporter vectors harboring a HIV-1 PRF signal
Increasingly, multimedia and video are being used in addition to traditional delivery methods to communicate scientific findings. Journals such as PLoS ONE, Cell, Nature and others often use video to either showcase particular articles, or offer authors the opportunity to include multimedia elements as part of their article. Some subjects lend themselves better to video delivery than others. Every so often a video report comes along that perfectly complements the content of the associated paper, illustrating the power of video to enhance communication of research findings.
In my opinion, the effective use of video to highlight results is beautifully illustrated by the report below, highlighting the publication “A synchronized quorum of genetic clocks” by Danino et al, which was published in Nature this week.