Blog Post 2: Inflammatory Lipids: Differences in Male and Female Production

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.

Blog Post 1- Inflammatory Lipids: Differences in Male and Female Production

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.