Working with samples in the lab in preparation to run rtPCR
Author: Breanna Williams | Major: Biology
Hi! My name is Breanna Williams, and I am a senior biology major in the Fulbright College of Arts and Sciences. In the fall of 2020, I began working in the Muscle Metabolism/ Cancer Cachexia Lab under the direction of my honors mentor, Dr. Tyrone Washington in the Human, Health Performance, and Recreation department. This fall (fall 2021), I began working on my honors thesis which is looking at how a leucine supplementation affects the quality of the mitochondria in animals that have colon cancer. Previous data indicates that leucine, a branched chain amino acid, promotes muscle synthesis, which explains why it’s often found in protein powders used by weight builders. However, data showing the effects of leucine in cancer patients is lacking. So, we hypothesized that a leucine supplementation would promote muscle synthesis in cancer mice and are in the process of testing that. We are using mice that have been specifically bred to have a mutant allele in the APC gene called APCMin/+. This mutation converts the gene into an oncogene and causes the mice to develop colon cancer, which is forms as little tumors, called polyps, in the mice’s colons.
When I started working in Dr. Washington’s lab, I was on the path to go to physical therapy school and found research with muscles not only interesting, but very applicable to my future career. I looked up the different labs on campus, liked the Muscle Metabolism Lab, and reached out to Dr. Washington explaining my story and desire to be a part of the lab. Not long into the semester, though, I realized that medical school was actually where I wanted to end up, but I still wanted to keep my focus on the musculoskeletal system. I learned about rehabilitation medicine and had been working with some PhD students on different experiments looking at muscle wasting in mice as a result of cancer. I knew I wanted my thesis to involve cancer research and its effects on muscles. When Dr. Washington told me that our lab didn’t have any data detailing the effects of cancer on the mitochondria in muscle cells and that I could take up that project, I knew that’s exactly what I wanted my thesis to be on.
Cancer is the second leading cause of death, and one key symptom of cancer is the wasting of muscle mass, also known as cancer cachexia. Slowing, and potentially reversing, this muscle wasting would help individuals with cancer to have a better quality of life. This research could identify a treatment that could slow this muscle wasting. As a result, this could be a key aspect in helping patients stay physically strong enough to better handle chemotherapy and promote their recovery.
I am working with a PhD student and a master’s student on this project and we are currently in the process of weaning the offspring, genotyping them, separating them into the experimental groups, and starting them on their designated experimental course. Genotyping is done by taking a small tail snip and ear punch off of each of the mice, placing those samples in a solution of forward and reverse primers plus a tagging agent, and then running the samples through an accelerated PCR to determine if the mouse has the APCMin/+ gene or not. The weaned offspring are weighed once a week, as well as their food and water, to document how much they are consuming and ensure that confounding variables are eliminated. We also perform grip strength testing for each mouse, each week, over the course of the experiment as another measurement to see how the muscle in the mice is being affected.
As the mice are genotyped, we separate then into 2 further subgroups: leucine supplementation and no leucine supplementation. We start them on their specific protocol (receive leucine enriched water or regular tap water) as soon as they are separated. The mice are also designated as either 13 week or 20 week mice. At the end of the mice’s designated experimental period, they will be anesthetized and have electrophysiology performed on them to determine their major muscle function. Then they are euthanized, and tissue samples of muscles and organs will be harvested and frozen for later analysis.
Though it’s still early in the process, we’ve faced a few challenges along the way. For starters, the mice breeders that we are using took later than expected to arrive, and because of some other setbacks, this APC project was slightly delayed, so the data collection and analysis won’t happen until next semester. We also discovered that our genotyping method was inaccurate and had to correct that before we could genotype the rest of the offspring. We don’t want any offspring to be mis-genotyped and placed in the wrong experimental group as that could skew results. But we are on track now and it is progressing well.
By the beginning of next semester, all the mice will be several weeks into their experimental protocol. We hope to have tissue harvests by early spring where we’ll collect MitoRespiration data to visualize the mitochondrial function of the mice, and then we will analyze the tissue samples and see if our predictions hold true. This Honors Grant has helped me get my thesis experiment started in a topic that I love, and I think will positively impact my future career as well as the future of cancer care. Throughout this semester, I’ve learned how to be more patient, as this isn’t something that can be rushed. I have also learned how to be more confident in myself in the lab and be okay when I make mistakes because I can use it as a learning experience and always improve the next time around. I’m thrilled to be able to continue this project and am thankful to have this grant to further support this research next semester.