Daniel Maxenberger

Author: Daniel Maxenberger | Major: Biomedical Engineering | Semester: Fall 2022

Over the course of the 2022 Fall 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 needed to restart the cloning process for the project. After many failed attempts, I redirected myself to then start optimizing and improving the process of cloning guides into a lentiviral backbone. This was a painstaking process as the lentiviral digestion required incubation times varying between 8 and 16 hours. This was before I ran the gel to verify successful digestion as well which takes another hour on top of this time. This meant that for the digestion alone it took at least an entire day from start to finish.

This has been a lengthy process as I have also had to go through the transformation protocol while optimizing this digestion. However, I am close to finishing the optimization of the protocol and can continue on with the guides successfully cloned into the plasmid soon.

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 will allow me to continue on to in vitro sooner rather than later. Also, if issues arise with this plasmid as well I fully intend on doing transient transfection to ensure my ability to proceed with in vitro testing

Upon returning to the lab in January, I have a few tasks to undertake. First I will continue the cloning protocol optimization for lentivirus while I also clone my guides into the new and less troublesome plasmid. I will then transfect Raw 264.7 cells with the guides and culture them so that I can perform an ELISA to check the effect transfection had on their state. This will provide data as to whether the transfection affected the cells and what changes there are in the state of the cells’ polarization. If able, I will compare transfection with both plasmids and transient transfection to see if the transfection mechanism has any effect.

This Honors College Research Grant allowed me to focus on working on my Honors Thesis more consistently. I love the subject my project revolves around, and the opportunity to do hands-on research I am passionate about has been phenomenal. The project has never failed to interest me, and the more I work on it the more respect and love I have for research. I am excited to see where this goes over my next and final.