At the Heller Lab of Neuroepigenetics, I have been working on a core project: What role does the cyclic dependent kinase 5 (CDK5) play in stress and memory? To do so, we first employ a variety of paradigms that include the Chronic Unpredictable (CUS) model and the Fear Conditioning (FC) paradigm.
In order to investigate epigenetic gene expression levels from these two paradigms, we extract RNA directly from brain tissue and then qPCR these samples. The qPCR is able to ascertain the copies of genes present after primers are able to cut at the specific sites. GAPDH is the gene used as the baseline control measurement, because of its ubiquity in cellular respiration processes. All qPCR values are normalized to the GAPDH counts.
After validating that there is a change observed from the CUS and FC protocols, mice undergo surgery with zinc finger protein (ZFP) or CRISPR- dCAS 9 primed viruses. These serve as mechanism to directly regulate gene expression in vivo. Thus, I have also assisted in experiments validating the potency of these viruses, with the hopes of conducting behavioral experimentation for stress and memory, if the viruses are deemed viable. These include forced swim tests (FST) to ascertain the depressive state of the mice after CUS.
While investigating the transcriptional mechanisms of CDK5 is a fundamental goal of understanding, studying the translation and post translational modifications to the epigenome are also of note. I have been running SDS Page Gels for solubilized protein samples from different brain regions of the stressed or fear conditioned mice. After, I transfer the gels to a membrane and select specific antibodies for the CDK5 protein of interest, I image the resultant using chemiluminescence. This Western Blot image is analyzed by the depth and width of the marks that are produced.
Once regulation of expression is deemed significant by the CDK5 gene in either CUS or FC paradigms, future experiments would entail more surgeries and observing behavior and epigenetic changes. For instance, for the CUS paradigm, we would determine whether mice have taken up stress “susceptible” or “resilient” phenotypes. This is the next step on the pathway from the molecular underpinnings of stress, to the phenotypes observed in traditional psychiatry.
I have truly felt my role in the lab gain more importance as the summer went on. After acquiring the necessary molecular and biochemical skills, members of the lab would frequently ask to run tests in their experiments. The first summer in the PURM program has enabled me to learn what it means to be a productive member in the lab, and how others will tend to rely on you. I am grateful to this program for fully submerging me into a field of science that I have grown passionate about, and in which I hope to make my own mark in the years to come.