Author: Daniel Maxenberger | Major: Biomedical Engineering | Semester: Spring 2023

Over the course of the 2023 Spring grant term, I continued to work in the lab of my honors mentor, Dr. Christopher Nelson, researching the possible polarization of macrophages using CRISPR gene editing to eventually be used as a potential therapy for diabetic wound healing complications which is the continuation of the project I have been working on since the beginning of the grant term in the Fall of 2021. Macrophages are a class of cells that can be widely divided into two phases: M1 and M2. M1 cells are pro-inflammatory cells that activate to clear out harmful bacteria and debris from wound sites by irritating it. After M1 cells activate and fulfill their role, they are polarized into their M2 phase. M2 cells are anti-inflammatory cells that begin the closing and healing process at the wound site.

The reason healing can be problematic for those with diabetes is that there is a delay between the polarization between the M1 phase and the M2 phase. This keeps the wound from closing properly and causes further inflammation delaying the wound closure process and allowing for the beginning of chronic wounds. The goal of our project is to use CRISPR gene editing to alter the malfunctioning macrophages to allow them to properly polarize into the M2 state.

This semester, I worked on optimization of cloning into the lentivirus plasmid for use in this project as well as others. This required extensive testing in all stages of transformation including digestion, ligation, and phosphorylation. Each step was optimized for both the time period that the materials needed to digest and ligate while also optimizing the desired concentration to allow for transformation and plating to be successful. This is what took a majority of the testing time. For the different time periods that the plasmid was digested at 55°C, nine different times were tested based on differing established protocols. For the number of differences in ligation, over 10 different ratios of insert to vector were tested before being able to determine the optimal concentration.  While optimizing the protocol I have also attempted to clone the guides into another plasmid as well that is not as difficult to work with as the lentivirus plasmid.

This semester has been largely focused on furthering the optimization of this protocol and writing my Honor’s Thesis. This research has thus allowed me to successfully create and present my Honor’s Thesis this past April and subsequently graduate with Honors in this past May.

Through the Honors College Research Grant, I have been able to more heavily pursue work on my Honor’s Thesis, and it allows for me to do it in a field and project that I love. Through my time in Dr. Nelson’s lab, my respect and admiration for research has continuously grown, allowing me to consider the role I would like research to hold for me professionally and has further encouraged me to pursue a PhD in Biomedical Engineering. The hands-on experience I have received has been invaluable with this and has only served to further foster my passion for the topic. The Honors College Research Grant has also been able to make me feel more comfortable devoting more time to my research than I normally would feel able to with the funding and has allowed me to successfully complete my Honor’s Thesis. Overall, the grant has provided me with a phenomenal opportunity that has allowed me to grow both professionally and academically, instilling in me a love of research that I hope to one day bring to my professional field and use in my future.