Research Department: Molecular Biology Graduation Date: December 2018
Abstract: The Kelch (KLHL) family of genes plays a role in protein ubiquitination, trafficking, and intracellular signaling. Many Kelch family members are associated with E3 ligase/ Cullin-RING ubiquitin ligase (CRLs) complexes and have been identified as playing roles in skeletal muscle organization and function. A recent study identified Klhl4 as expressed in endothelial cells using unbiased single cell sequencing of mouse heart during cardiac development. These data suggest that Klhl4 may be associated with vascular development. To characterize the expression of Klhl4 in adult murine tissues, we performed RT-qPCR using a multi-tissue library, and found that Klhl4 was most highly expressed in brain, ventricle, atria, and adipose tissues. It was also more highly expressed in non-cardiomyocytes compared to isolated cardiomyocytes. To determine where Klhl4 localizes in vitro, we overexpressed a C-terminus FLAG-tag Klhl4 construct and transfected our vector in Cos-7 cells where we observed ubiquitous expression of Klhl4 throughout the cytoplasm. In contrast, N-terminus FLAG-tagged Klhl4 appeared to localize to fiber-like structures in C2C12 cells. In order to determine the in vivo role of Klhl4 we generated a knockout mouse using the Clustered Regularly Interspaced Short Palindromic Repeats/Cas-9 system (CRISPR/Cas-9) with a single-guide RNA (sgRNA) targeting exon 4 of Klhl4. We confirmed induction of indels at the Klhl4 locus by genotyping of our founder mice. Future studies will focus on generating and characterizing a complete Klhl4 knockout mouse to elucidate its in vivo role with a focus on vascular development and homeostasis.
"Green Fellows has taught me that the beauty of research is concealed in the pursuit of analytical thoughts to answer a fundamental question rather than the accumulation of fluency in performing tandem techniques related to your field of study."
What does research mean to you? My passion in research has always been in developmental biology– specifically in cardiac and muscle development. I have found that your interests in an unanswered question often leads to your pursuit of that topic in research. For me, cardiology was something that hits very close to home, so the Green Fellows had allowed me to learn as much as I can about the mechanisms of heart regeneration. The opportunities in the Olson lab have allowed for a palate of interest in the characterization of a novel protein gene that is robustly expressed in endothelial specific tissues. As a chemistry major, it was rewarding to go through the transition towards a very intense focus on molecular biology laboratory techniques. Green Fellows has taught me that the beauty of research is concealed in the pursuit of analytical thoughts rather than the accumulation of fluency in performing tandem techniques related to your field of study. Ultimately, my satisfaction from Green Fellows has been the progression of independence in thoroughly understanding the purpose behind many molecular techniques to ask and answer fundamental questions that pertained to my project.
Tell us about your journey. I was very overjoyed to have been accepted into such a prestigious program. At the orientation, I had already met with my laboratory PI and discussed the atmosphere and my project over the lab. I had little to no background in molecular biology research, so it was a sharp learning curve for me. Luckily, I was able to start a project with my post-doctorate fellow there.
Create your own unique website with customizable templates.