Ahmed Tolba

Author: Ahmed Tolba | Major: Biomedical Engineering | Semester: Spring 2024

During my freshman year, I took an honors college gene editing forum where Dr. Chris Nelson lectured on CRISPR applications, history, and ethics. His class sparked my interest. After the class, I emailed Dr. Nelson, expressing my enthusiasm for his research and asking if I could join his research group. He welcomed me, and I began working in his lab.

I learned that macrophages, a type of immune cell, can change their behavior based on environmental signals, a process called polarization that affects inflammation. Our goal was to eventually use the dCas9-KRAB system to control this process. dCas9-KRAB reduces gene activity without cutting DNA, allowing us to study how gene expression influences cell behavior. The potential applications of this technology are vast. For example, in patients with diabetes, chronic wounds are a significant complication. These wounds often fail to heal properly due to persistent inflammation and poor macrophage function. By using dCas9-KRAB to modulate gene expression in macrophages, we could potentially reduce inflammation and promote better wound healing. This approach could lead to new treatments that specifically target the cellular mechanisms underlying chronic wounds, improving outcomes for patients with diabetes and other conditions that affect wound healing.

One of the biggest challenges to this research was the molecular cloning process. Cloning the specific gRNAs, short RNA sequences that guide the system to specific locations in the genome to modify gene expression, for IL-6 and IL1β required careful planning and execution. There were several instances where the cloning didn’t work, leading to delays and frustration. Notably, several other genes aside from IL-6 and IL1β, such as TNF-α, also failed to clone.

Cloning genes into a lenti-guide backbone is a common method in genetic research, but several common issues can cause the process to fail. One common problem is incorrect sequence or design errors. Think of it like trying to assemble a complex piece of furniture with faulty instructions. If the manual has errors or the parts are mislabeled, the final product won’t come together correctly. Similarly, in genetic cloning, if there’s a mistake in the genetic sequences or the design of our constructs, the entire process can fail, preventing successful cloning. Another issue is low efficiency of ligation, where DNA pieces are joined together using enzymes, akin to gluing puzzle pieces. If the ligation conditions are suboptimal or the enzyme quality is poor, the DNA fragments won’t stick together properly. Additionally, restriction enzyme issues can arise. These enzymes act like precise scissors, cutting DNA at specific spots. If the enzymes don’t cut accurately due to improper conditions or degraded enzymes, the DNA pieces won’t fit together, much like trying to connect pieces of a jigsaw puzzle with mismatched edges.

Finally, transformation efficiency is an important step where the recombinant DNA is introduced into bacterial cells, similar to trying to send an important message via email. If the internet connection is weak or the email server is down, the message won’t be delivered. In the lab, if the bacterial cells aren’t healthy or the conditions for introducing the DNA aren’t optimal, the DNA won’t be taken up by the cells, leading to poor cloning results. Understanding these potential issues helps researchers troubleshoot and refine their methods to achieve successful cloning in genetic research.

Balancing coursework and research responsibilities was another challenge. Strong organizational skills were necessary. I sought guidance from my PhD student mentor and recent UofA graduate, Dr. Allie Ivy, who helped refine protocols and boost my skills and confidence. For time management, I created a schedule and prioritized tasks to ensure I could dedicate sufficient time to both my studies and research. Dr. Nelson’s guidance was instrumental in shaping the project and overcoming technical challenges. I worked closely with fellow lab members, sharing knowledge and troubleshooting experiments together, which accelerated our progress and taught me the value of collaboration in science.

I presented my research at the National Undergraduate Research Week Symposium. Presenting allowed me to share our findings, receive feedback, and network with other student researchers, enhancing my communication skills and confidence. The next steps involve further validating our cell line and cloned gRNAs. Future experiments will focus on introducing these gRNAs into the macrophage cell line and assessing their ability to control gene expression. This work will help us understand how this system can influence macrophage behavior and its potential as a therapeutic tool. Personally, this research experience has reinforced my desire to pursue a career that combines patient care and scientific discovery. After completing my undergraduate studies, I plan to attend medical school. The skills and knowledge I’ve gained have prepared me for future challenges and inspired me to contribute to biomedical research and healthcare.