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.
Our research focuses on inflammatory lipid production in mice with a mutated COX2 enzyme, but we are currently in the preliminary stages. In our research, thus far, we have focused on genotyping each litter of mice in order to determine in which cases we were successful in the mutation of the gene. Last semester we finished writing a procedure for the process and at this point, have successfully performed two rounds of genotyping. We have experienced some difficulties in this process. The gel for the first litter we attempted to genotype did not turn out. We then performed gel electrophoresis and PCR again, in attempts to determine where we went wrong in the process. Based on the fact that the second gel produced results after a repeat of PCR, we believe that the error occurred at that step in the process. From that point on we changed the procedure in regards to PCR. We reworked the procedure to include mixing at each step in the PCR process and no longer leave the PCR products over night in the machine to prevent issues within the gel electrophoresis. We also recently experienced issues with our shaking water bath, which is used for DNA extraction from the tail tips. During our most recent round of genotyping, the machine experienced difficulty maintaining a constant shaking rhythm, which is necessary for the DNA extraction. We believe this could have happened due to age or a lack of use. Despite these issues, we were able to successfully genotype our current litter.
This process has been extremely helpful to me in gaining knowledge of what research actually looks like. I’ve almost enjoyed some of the setbacks we’ve had if only because it gave me experience problem solving when our experiment didn’t produce viable results. It can be extremely difficult pinpointing the spots in your research were you’ve gone wrong and I’ve never actually had to go through this process in-depth to fix issues that arise in lab. This process has also helped me gain an understanding of an in-depth research process. Although we have a big picture research question, we have only been able to take the first steps toward this process. While it may seem a small part, the genotyping is a crucial step in the whole process. We cannot start to look at prostaglandin production until we know which mice are actually usable in our study. Lastly, I have enjoyed so much, working with and learning from Dr. Upmacis. She has been such a great mentor, including me in the process of problem solving when issues arise.
So far in our research we have written a procedure for genotyping and successfully genotyped one of the litters. We worked off of a previously written protocol and adjusted things as were necessary, adding in more information about locations of reagents and what adjustments we had to make along the process. We performed PCR and gel electrophoresis, twice before it was successful because the gel did not turn out the first time. The first gel produced bands all at the same band length, which indicated that something went wrong in the process. We knew that the error did not occur in the process of running the gel itself because we did get bands and the DNA ladder turned out as well. We believed there was an issue with our PCR (polymerase-chain reaction) product which resulted in this error. The PCR product could have sat for too long before being used or it was not mixed well enough before use. We repeated the process, making sure to be extra careful with our technique this time, especially with pipetting and mixing the PCR products that we generated. This resulted in a successful gel. We then analyzed the gel to determine the genotypes of each mouse and whether they were wild-type (WT), heterozygous, or homozygous for our gene. We have also started writing up another protocol using a previously published protocol for protein calibration assays which we hope to start in the spring semester.
I didn’t know much about the project when we first started and Dr. Upmacis was very helpful in showing me some of the literature and explaining some of the theory behind our project. We worked together in updating the previously written procedure and she has provided me with supervision when working on the gel. This process has required a lot of communication between the two of us, both to figure out times we are both available to work and to problem solve issues that arise. Throughout our research, I have learned how to perform the process of PCR, which I had never done before. I’ve also learned more about making necessary adjustments when experiments are unsuccessful.
Our research project was initially focused on the effects of consumer products on the health of cells. However, we recently had to change our project due to issues with instruments necessary to complete the research. The research has now shifted to looking at the differences in lipid production of male and female mice with a specific mutation. The title of our new project is “Inflammatory Lipids: Differences in Male and Female Production.” In this research we are looking specifically at lipid production of both male and female mice that have been genetically modified to produce a mutated COX2 enzyme that has a phenylalanine amino acid instead of a tyrosine amino acid at position 385 of the enzyme sequence (COX2Y385F). This mutation causes a loss of function in the protein which functions in the production of prostaglandins. Prostaglandins are lipids that aid in the regulation of pathogenic mechanisms such as inflammatory response. Due to its function in the inflammatory response, the COX enzyme has been a target for pain reliever medications such as aspirin and ibuprofen.
Initial clinical trials that provided information for effects of pain reliever medications included only men. Furthermore, until recently, medical research using rodents tended to use only males, due to the difficulty and expenses in studying both sexes. This research will help determine if there are any previously unknown differences in inhibition of the COX enzyme based on sex. We will first genotype the various mice to determine which mice have the mutation and which will serve as controls. This step will involve DNA extraction, PCR of extracted DNA, and gel electrophoresis. In the last few weeks, I have mastered the skills involved in genotyping and I have helped to develop a detailed protocol. Next we will determine the lipid production of these mice by analyzing the urine and protein concentration using a protein assay. These data will aid in determining differences in female and male production of prostaglandins with inhibited COX2 enzyme.