Our research centered on the possibility of differences in inflammatory lipid production between male and female mice with a COX2 partial mutation from a tyrosine to phenylalanine at position 385 in the enzyme sequence (COX2Y385F). This mutation causes a loss of function in the cyclooxygenase (COX) pathway, which inhibits the production of inflammatory lipids or prostaglandins. These are lipids which aid in the regulation of the body’s inflammatory response, as well as other pathogenic mechanisms. Due to its function in the production of prostaglandins this pathway has been the target for anti-inflammatory medications like aspirin and ibuprofen, to treat the concurrent symptoms. Initial research and clinical trials on the effects of such medications were only done with men, and there has been little to no research on the whether there are differences in the way they affect women. This research will focus on filling in those gaps of information in the current research to determine if there are differences in the production of prostaglandins between males and females. We focused mainly on genotyping the mice, which is the first crucial step to this study. Over the past year we have developed a protocol to determine which mice have the mutation.
The genotyping protocol which we developed involved four main steps. First the mice had the tips of their tails cut. These tail tips were then used to perform DNA extraction and then polymerase chain reactions (PCR) to amplify the extracted DNA. Amplification is necessary to make sure there is enough DNA to examine properly. Lastly the PCR samples are run through a gel, using gel electrophoresis. This process will generate a gel with bands specific to each genotype; wild-type with no mutation (WT), heterozygous with a partial mutation (HET), and knockout with a mutated gene (KO). In the beginning of our research we experienced issues with the outlined process and worked throughout the year to develop a more extensive, in-depth protocol. We think the issues we experienced were due to unmixed PCR samples. We have since added a stronger emphasis on mixing the PCR samples extensively before performing gel electrophoresis. This addition to the protocol has seemingly fixed our initial problems, as have since been able to run successful gels for three different litters. The first two gels generated only mice with WT and HET genotypes. On the third gel we were able to get one mouse with a KO genotype but the rest displayed HET or WT. These results indicate that the Mendelian genetics failed in our experiment, as these mice were bred from two heterozygous mice. This would indicate that about 25% of the next generation should have the KO genotype while on one has so far in three litters. It is important to obtain as many mice with the KO genotype as we can to complete our future experimentation, which will include urinary analysis of prostaglandins in mice with the mutated gene.
Overall this experience has been extremely rewarding for me, both in my research and laboratory experience. Dr. Upmacis has been an incredible mentor to me, including me in the process of developing our protocol from the very beginning. I cannot say how valuable that was to me, as it helped me see and participate in the process of generating a research protocol. Classes and even lab can be so isolating because we never actually see the process of adapting to circumstances by implicating changes in the protocols that we use, so this experience gave me a deeper insight to what I will be doing once I graduate. Problem solving when issues arise is a crucial component to research, so experiencing these challenges has given me more insight to apply the knowledge that I’ve gained from my classes. I also gained exposure and experience with the laboratory techniques of DNA extraction, PCR, and gel electrophoresis. Before starting on this project I had no experience with the first two steps and very minimal experience with the process of gel electrophoresis. This project has expanded my repertoire in lab which will only help me in the future.