CRISPR/Cas9 HiBiT Knock-In: A Scalable Approach for Studying Endogenous Protein Dynamics

Posted on by Darcia Schweitzer

Studying protein function in live cells is limited by the tools available to analyze the expression and interactions of those proteins. Although mass spectrometry and antibody-based protein detection are valuable technologies for protein analysis, both methods have drawbacks that limit the range of targets and contexts in which proteins can be investigated.

Mass spectrometry is often poor at detecting low-abundance proteins. Antibody-based techniques require high quality, specific antibodies, which can be difficult to impossible to acquire. Both methods require cell lysis, preventing real-time analysis and limiting the physiological relevance, and both methods can be limiting for higher-throughput analysis. While plasmid-based overexpression of tagged target proteins simplifies detection and can allow for real time analysis, protein levels don’t typically resemble endogenous levels. Overexpression also has the potential to create experimental artifacts or limit the dynamic range of an observed response.

In 2018, Promega R&D scientists published a paper in ACS Chemical Biology demonstrating the use of CRISPR/Cas9 to integrate the 11 amino acid, bioluminescent HiBiT tag directly into the genome to serve as an easily measured reporter for endogenous proteins. This provides a highly quantitative method for investigating cellular protein dynamics that sidesteps the need for cloning and other drawbacks to conventional methods, including the ability to measure changing protein dynamics in real-time. (For more details about CRISPR/Cas9 knock-in tagging and other applications, read this blog.)

While their findings showed that this method provides efficient and specific tagging of endogenous proteins, the research was limited to just five different proteins within a single signaling pathway in two cell lines. This left unanswered questions about whether this approach was scalable, had broader applications and how accurately the natural biology of the cells was represented.

Continue reading “CRISPR/Cas9 HiBiT Knock-In: A Scalable Approach for Studying Endogenous Protein Dynamics”

In Vitro Transcription and the Use of Modified Nucleotides

Posted on by Leah Cronan
In vitro transcription
RNA polymerase unwinds DNA strands for transcription.

Transcription is the production of RNA from a DNA sequence. It’s a necessary life process in most cells. Transcription performed in vitro is also a valuable technique for research applications—from gene expression studies to the development of RNA virus vaccines.

During transcription, the DNA sequence is read by RNA polymerase to produce a complimentary, antiparallel RNA strand. This RNA strand is called a primary transcript, often referred to as an RNA transcript. In vitro transcription is a convenient method for generating RNA in a controlled environment outside of a cell.

In vitro transcription offers flexibility when choosing a DNA template, with a few requirements. The template must be purified, linear, and include a double stranded promoter region. Acceptable template types are plasmids or cloning vectors, PCR products, synthetic oligos (oligonucleotides), and cDNA (complimentary DNA). 

In vitro transcription is used for production of large amounts of RNA transcripts for use in many applications including gene expression studies, RNA interference studies (RNAi), generation of guide RNA (gRNA) for use in CRISPR, creation of RNA standards for quantification of results in reverse-transcription quantitative PCR (RT-qPCR), studies of RNA structure and function, labeling of RNA probes for blotting and hybridization or for RNA:protein interaction studies, and preparation of specific cDNA libraries, just to name a few!

In vitro transcription can also be applied in general virology to study the effects of an RNA virus on a cell or an organism, and in development and production of RNA therapeutics and RNA virus vaccines. The large quantity of viral RNA produced through in vitro transcription can be used as inoculation material for viral infection studies. Viral mRNA transcripts, typically coding for a disease-specific antigen, can be quickly created through in vitro transcription, and used in the production of vaccines and therapeutics.

Continue reading “In Vitro Transcription and the Use of Modified Nucleotides”

Designing BET(ter) Inhibitors to Guide Therapy for Cancer and Inflammatory Diseases

Posted on by Ken Doyle
bet proteins brd nanoluc

Transcriptional activation of genes within the nucleus of eukaryotic cells occurs by a variety of mechanisms. Typically, these mechanisms rely on the interaction of regulatory proteins (transcriptional activators or repressors) with specific DNA sequences that control gene expression. Upon DNA binding, regulatory proteins also interact with other proteins that are part of the RNA polymerase II transcriptional complex.

One type of transcriptional activation relies on inducing a conformational change in chromatin, the DNA-protein complex that makes up each chromosome within a cell. In a broad sense, “extended” or loosely wound chromatin is more accessible to transcription factors and can signify an actively transcribed gene. In contrast, “condensed” chromatin hinders access to transcription factors and is characteristic of a transcriptionally inactive state. Acetylation of lysine residues in histones—the primary constituents of the chromatin backbone—results in opening up the chromatin and consequent gene activation. Disruption of histone acetylation pathways is implicated in many types of cancer (1).

Continue reading “Designing BET(ter) Inhibitors to Guide Therapy for Cancer and Inflammatory Diseases”

How to Start Writing a Scientific Manuscript

Posted on by Promega

Today’s blog is adapted from a presentation by Danette Daniels, PhD, in our webinar “Writing About Science: Tips and Tricks for Communicating Your Research.”

As scientists, we can do science forever. The beauty about science is that the questions never end – we can keep asking, and every time we find an answer, we have a new direction to pursue. But it’s very important to know when it’s time to write up your results.

Publishing may be connected to leaving or transitioning your position, but at all times you should be thinking, “What is my end goal? What is the big question I want to answer? What are the questions the field has about my research?” As you reach milestones and make discoveries, whether big or small, consider whether you will have a complete and compelling story to tell in the end.

Continue reading “How to Start Writing a Scientific Manuscript”

Using molecular biology and biochemistry to detect blood doping

Posted on by Michele Arduengo

Monitoring the use of performance-enhancing substances among athletes is complex and the requirements for tests and assays that detect use of such substances have changed significantly over the last few decades.

The haematological (blood) module of Athlete Biological Passport was adopted December 1, 2009 (ABP) by the World Anti-Doping Agency. The module sets out standard protocols to monitor doping of professional athletes by looking at changes in biological parameters, without relying on the detection of illegal compounds in body fluids. Such biological methods eliminate the need to develop and validate a test to detect every new compound that can be used for doping. The current version of the ABP, adopted in 2014, also adds monitoring of certain steroid use indicators from urine samples.

Blood doping which aims at increasing red blood cells so that more oxygen can be transported to muscles to increase stamina or performance is particularly difficult to detect. There are typically three ways that it is accomplished: use of erythropoietin (EPO) or synthetic oxygen carriers and blood transfusions. While transfusions of large volumes of blood or use of EPO can be detected, microdosing EPO or transfusing smaller volumes of packed red blood cells is much harder to detect.

Nicolas Leuenberger and colleagues at the Swiss Laboratory for Doping Analysis have developed a method to detect blood doping. In addition to addressing the detection of blood doping, his laboratory is also concerned about easing the transport and storage requirements for samples and ensuring that sample collection does not adversely affect athlete performance.

Improving Collection and Storage of Blood Samples

Because sample collection and storage are so critical to accurate test results, any new assays developed to detect blood doping benefit from ease of collection and storage. The Leuenberger laboratory investigated the use of the TAP™ Push Button collection device, which is billed as a simple method for blood collection that is easy to use and eliminates the need for painful needle sticks or finger pricks that can affect athlete performance. After TAP collection, 20µl of blood from the device was placed on to filter paper and dried (dried blood samples; DBS), which are much easier to store and transport from collection site to laboratory.

An RNA Biomarker for Blood Doping

Blood withdrawal and autologous transfusion or recombinant human EPO injection stimulate erythropoiesis and immature red blood cells can be distinguished based on their gene expression profiles. One of the genes that is expressed by immature red blood cells is aminoleuvulinate synthase 2, a gene that encodes an enzyme ALAS2 involved in the synthesis of heme, a pathway active during RBC maturation. RNA transcripts are unstable and tend to degrade rapidly, so isolating linear RNA transcripts from a collected sample can be difficult. However circular RNAs (circRNAs) are a class of RNA molecule produced by the backsplicing of pre-mRNAs that are high in abundance, quite stable and maintain cell-type specific expression. The Leuenberger laboratory developed a method for measuring the linear and circular forms of ALAS2 RNA in DBS to monitor erythropoiesis.

One of the greatest challenges in developing this protocol was achieving efficient RNA extraction from only 20ul of dried blood. Leuenberger and his colleagues adopted a two-step purification; beginning with a phenol:chloroform extraction on the DBS followed by a further purification on the Maxwell® RSC automated instrument, using the Maxwell RSC miRNA Serum and Plasma kit. Switching from a manual to an automated method for the second step was crucial. It reduced chances of contamination as well reduced pipetting errors, without compromising good quality and yield of RNA therefore contributing to assay reproducibility. To normalize volumes within the blood spot, the protocol uses RNA produced by housekeeping genes. The work to automate the assay has been published in Bioanalysis.

What’s Next

This protocol is being tested to see if microdosing of EPO or small transfusions can also be detected by monitoring ALAS2 RNA expression in DBS. The Swiss laboratory of Doping Analysis is also in the process of developing a method to detect gene doping by isolating plasmid DNA from whole blood samples, using the Maxwell® RSC.

Additionally, the collection and storage methods used have implications for the clinic, especially for patients that need routine blood monitoring. The ability to isolate circular RNAs shows promise in forensic applications to identify body fluids. 

Want to know more about how the Maxwell® RSC can give you the freedom to focus on the work that interests you the most? To learn more, click here.

Uncovering the Origins of the Commensal House Mouse

Posted on by Sara Klink
Figure of house mouse. Copyright George Shuklin.
📷: George Shuklin

When I encounter my cat fixated on specific locations in my kitchen, her behavior shows me that she has heard some mice in those areas. In fact, mice have been attributed as a reason that cats became companions to humans. Mice start gathering and reproducing so cats followed the food source and hunted the rodents, thus endearing themselves to humans, who were storing food for their own use. However, new evidence described in Scientific Reports has shown that mice have been associated with humans even before grain storage was widespread. In fact, by making our dwellings comfortable, we also created an inviting place for mice to live.

Continue reading “Uncovering the Origins of the Commensal House Mouse”

NanoBRET™ Assays to Analyze Virus:Host Protein:Protein Interactions in Detail

Posted on by Kyle Hooper

Recently, Gordon et al. published an atlas of protein:protein interactions of all proposed SARS-CoV-2 proteins expressed individually in HEK 293 cells (Table 1). The study tagged each of the viral proteins with an epitope tag and performed a pull-down of the expressed protein followed by trypsin digestion and mass spec analysis, a process referred to as affinity purification–mass spec analysis. The group identified 332 human proteins interacting with 27 SARS-CoV-2 proteins.

The interactions identified in the HEK 293 cells helped Appelberg et al. analyze interactions over time in SARS-CoV-2-infected Huh7 cells. Gordon et al. used the PPI data to identify FDA-approved drugs, drugs in clinical trials, and pre-clinical compounds that bound to the identified human proteins and labs in New York and Paris tested some of these drugs for antiviral effects.   

Table 1. The general functional area of human proteins identified to interact with individually expressed SARS-CoV-2 proteins as reported by Gordon, et al. (1). The SARS-CoV-2 proteins are classified as non-structural proteins (nsp#), structural proteins (E, M, and N) and accessory proteins (orf#).  
Continue reading “NanoBRET™ Assays to Analyze Virus:Host Protein:Protein Interactions in Detail”

Using Wastewater Surveillance to Track COVID-19 Outbreaks

Posted on by Promega

Today’s blog is written by guest blogger, Sameer Moorji, Director, Applied Markets.

Even as countries are now gradually starting to reopen after lockdown, the COVID-19 pandemic is far from over. Researchers around the world continue to find new ways to monitor, prevent and treat the disease. One new way of monitoring COVID-19 outbreaks relies on a somewhat unexpected source: sewage water.

In March 2020, researchers at the KWR Water Research Institute found the presence of SARS CoV-2 RNA in wastewater samples collected near Schiphol airport in Amsterdam and several other sites in Netherlands. The result came within a week after the first case of COVID-19 in the country was confirmed. This study opened the door to the possibility of using wastewater-based epidemiology to determine population-wide infections of COVID-19.

What is Wastewater-based epidemiology?

Wastewater based epidemiology (WBE), or sewershed surveillance, is an approach using analysis of wastewater to identify presence of biologicals or chemicals relevant for public health monitoring. WBE is not new, as wastewater has previously been used to detect the presence of pharmaceutical or industrial waste, drug entities (including opioid abuse), viruses and potential emergence of super bugs. In fact, several countries have been successful in containing Polio and Hepatitis A outbreaks within their geographic locations.

Continue reading “Using Wastewater Surveillance to Track COVID-19 Outbreaks”

Promega Leverages Long-Time Experience in MSI Detection with European Launch of CE-Marked IVD Assay for Microsatellite Instability

Posted on by Kelly Grooms

The genetic abnormality called microsatellite instability, or MSI, has been linked to cancer since its discovery in 1993 (1). MSI is the accumulation of insertion or deletion errors at microsatellite repeat sequences in cancer cells and results from a functional deficiency within one or more major DNA mismatch repair proteins (dMMR).  This deficiency, and the resulting genetic instability, is closely related to the carcinogenicity of tumors (2).

Historically MSI has been used to screen for Lynch Syndrome, a dominant hereditary cancer propensity. More recently, tumors with deficient MMR function have been identified as being more likely to respond to immune checkpoint inhibitor (ICI) therapies (3.). Because MSI can be the first evidence of an MMR deficiency, MSI-High status is predictive of a positive response to immunotherapies such as ICI therapies. (3).

Learn more about MSI in this short animation.
Continue reading “Promega Leverages Long-Time Experience in MSI Detection with European Launch of CE-Marked IVD Assay for Microsatellite Instability”