Performing Microscopic Analysis post Lipofectamine Transfection
Author: Ryan Rouse | Major: Biomedical Engineering | Semester: Spring 2025
Ryan Rouse is conducting this biomedical engineering research under the guidance of Dr. Christopher Nelson within the College of Engineering during the 2024-2025 academic year. Ryan Rouse will graduate in the spring of 2025 with a Bachelor of Science in biomedical engineering. After graduation, Ryan will attend medical school at UAMS in Little Rock, AR.
Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder that primarily affects boys. DMD causes a significant reduction in life expectancy due to progressive muscle deterioration through a mutation in the dystrophin protein. Gene editing techniques have been established as a way to combat the effects of DMD and have gradually shown progress in increasing the life expectancy for DMD individuals. A new gene editing technology, CRISPR-DREAM, has the potential to revolutionize CRISPR technologies through its use of human mechanosensitive transcription factors as transactivation domains. This research focuses on achieving optimal activation of CRISPR-DREAM in C2C12 mouse muscle cells so that it can be used in further studies to upregulate proteins similar to dystrophin. If CRISPR-DREAM proves practical by upregulating replacement proteins for dystrophin, it can be explored as a gene therapy technology for Duchenne muscular dystrophy patients.
Towards the end of my junior year, I joined Dr. Nelson’s lab on his “DREAM Team.” This team consisted of two undergraduates and one graduate student working on different aspects of the same project. My fellow undergraduate teammate was already well on his way to completing his work once I joined the team. It was immensely helpful learning from a peer who was already acclimated to the lab. I was able to initially observe and learn before finishing his work, as he was studying abroad. While I was finishing his work last semester (Fall of 2024), many obstacles arose and caused our team to pivot and adapt our work. This caused my entire thesis focus to shift, and this was my first exposure to the trials and tribulations that come with biomedical engineering lab work. Engineers are no strangers to problem-solving, and our team was determined to find an answer to the problems arising in our DREAM experiments.
This brings me to my final semester of research, which was filled with trial-and-error experiments. A major issue we faced was finding an optimal delivery vector for the CRISPR-DREAM sequence into the cells we were testing on. Me and my fellow “DREAM Team” member, Daniel Maxenberger, set out to compare two different modes of delivery in order to uncover which method was preferred by the DREAM system. Though I had originally planned on designing a different experiment for my thesis, this comparison of delivery methods became my thesis focus. I have found this is a common experience in the lab. One test answers one question but causes more questions that need to be answered to arise (similar to the Hydra’s heads from Greek mythology). Through many tests and many failures, we finished the semester with promising data and progress in using CRISPR-DREAM as a potential gene therapy technology for muscular dystrophy.
My research journey was full of talented, hard-working superiors who were never shy to lend me a helpful hand. First off, Dr. Nelson was everything an undergraduate researcher could hope for in a research mentor. He was always willing to spend time discussing lab procedures, failures, and redesigns with me. He was more than just a research mentor, though; he became someone I looked up to in all facets of life. His work ethic and personality rubbed off on me and were a key to my success in the lab. I am immensely grateful for his mentorship. Similarly, Dr. Nelson’s graduate students, Shilpi Agrawal and Abbey Stokes, became extensions of his mentorship to me. They were quick to answer my arising questions that came from conducting experiments. They motivated me to continue my work through many failures and made me believe I was capable of completing what I set out to do. Research was no different than my experience with sports teams; people with a common goal coming together and sacrificing time and effort to reach that goal.