Dr. Arturo Diaz, PhD

My name is Arturo Diaz and I'm an assistant professor at La Sierra University. My lab works on brome mosaic virus which is a plant virus that can replicate in the yeast Saccharomyces cerevisiae. And we use it as a model system to study some of the common features shared by all positive-strand RNA viruses.
And there are many human pathogens that belong to this category, including hepatitis C, Dengue, SARS, and the recently emerged Zika virus. We might be able to design broad-spectrum antiviral drugs that target not just one virus, but multiple viruses that belong to this category. Some of the common techniques that we use in the lab involve measuring gene expression, protein levels as well as the determining the localization of cellular proteins in the absence versus the presence of the virus.
One of our go-to assays in the lab is measuring viral RNA levels and cells in which specific genes have been knocked out in order to determine the contribution of those genes to viral replication. So that process normally involves the extraction of total RNA from the cells. We take that RNA and we convert it into cDNA.
You've seen the reverse transcript is super mix. We then take that cDNA and using the SsoAdvanced SYBR Green mix, we essentially measure RNA levels using the CFX Connect Real-Time PCR System. And this not only allows us to measure viral replication or viral RNA levels within the cell but also measure particular transcripts which allows us to normalize our results at the end of the assay.
One of the advantages of using the CFX Connect System is that it's very user-friendly and this is of importance to me because my lab consists exclusively of undergraduate students who don't have really that much research experience and so I'm able to train them on how to use the software within 10 minutes.
We can get reproducible data with volumes as low as 10 microliters per reaction which minimizes the amount of sample and reagents that we have to use. One of the advantages of using the iScript Supermix is that all the reagents come in a single tube which reduces the chances of human error when you're trying to put all the reactions together.
When setting up our qPCR reactions, we use the SsoAdvance SYBR Green Supermix. We do various different assays and we're able to use the same reagents. This is advantageous because we have a lot of undergraduate students and so it reduces the possibility that they'll forget that they added a certain component and it also increases our reproducibility going forward because again, there's less pipetting errors during our experimental setup.
Going forward, our lab is interested in identifying and characterizing other host factors that are required to form and maintain these member inbound replication compartments. One of the big aspects that we're interested in is trying to determine how are the membranes being invaginated and how does high curvature maintain since it's energetically unfavorable.
Within the field of positive-strand RNA viruses, One of the most exciting developments has been the optimization of the CRISPR/Cas9 system and the reason for this is up to this point, people have had a really hard time knocking down genes and mammalian cells. With the CRISPR system, it's a lot easier to disrupt sailor function so that we can look at the contribution of specific genes to viral replication.
Using the CRISPR system, we could provide alternative antiviral therapies instead of using drugs like we currently are at the moment.