Beginning in early May I began working on my UV and melanoma research project. Because my research is based around how melanoma develops in mice who have two very specific mutations, these mice have to be made and bred to achieve this genotype. This is where I began with my project. We got the CRISPER mice from a different lab which had the gene for Albinism and then began to breed them with our current colony of mice that had the correct genes for the melanoma causing mutations, BRAF and NRAS. The results from these crosses yield mixed litters of black and white pups with different combinations of the genotypes. To determine which white mice had the correct genotype for our study we had to preform several PCR reactions with samples of their DNA to test for the three different criteria that we were looking for BRAF/NRAS mutant, loss of the tumor suppressor gene p16, and a positive result for Tyrosinase Cre. This process took up the entire month seeing as we had to wait for each litter to be born.
While I continued to breed the albino mice in June I was also focused on sequencing Tumor DNA from the black mice from the previous study conducted in my lab. This data will be compared to the data collected from the white mice, and therefore it was very important that it was analyzed. The first step I had to complete was analyzing each mutation found in each of the 20+ tumor samples. This took about a week and a half since the mutation burden was so high for these samples. After I had this complete I had to sort the mutations by the gene that they effected and then I had to design primers that would specifically target the exact spot on the genome. By designing these primers, we will be able to run sequencing on the tumor samples and this will allow us to validate the presence of the mutation. After a week I the primers came in and I was able to set up the sequencing for each tumor sample and submit them. After a few days I got the results back and I was able to analyze all of the sequencing results. I compared the results to the known sequence from the literature for each gene tested in each tumor sample. If the mutation was seen in the tumor sample, and it looked different than the normal gene sequence then the mutation was validated.
With the tumor validation done I could focus again on my breeding of the Albino mice. Which had progressed to the point where we had several white mice with the correct genotype who could then be back crossed to produce our pure line of mice that we could use for the study. We hit a road block in genotyping these mice when two of the three genotypes came out as expected but the p16 has been causing me issues. It appears that one of the mice was initially genotypes wrong and now some of its progeny are not showing the right gene expression, so this definitely counts as a setback to the project. Once we get these sorted out then we will know that we have a pure colony of albino mice and we can begin exposing them to UVA and B and monitoring them for tumors.
August Reflection (overall reflection)
This summer I was able to complete a lot to set up the foundations of my project for the upcoming year. In these three months I have been collecting the data on the black mice that have been exposed to the UVA and UVB and I have been breeding a colony of the white mice that we will soon begin to expose to UV. In the analysis of the black mice data I have had to check and harvest many tumors from our experimental mice. This tumor DNA was then analyzed for mutations and those mutations were organized and grouped. Specific PCR primers were developed to target the site of the mutation that was picked up in the analysis and the reaction with the DNA and the primer was sequenced. After sequencing the results were analyzed and most of the mutations originally seen were validated and confirmed.
I have also had a lot of success in preparing the albino mouse colony for the next stage of the project. After starting with only a few white mice and breeding them with our existing black mice to obtain our desired genotype, we have finally established at least one litter of the pure white strain with the correct genotypes. After they are of breeding age we can begin the process of painting the mice with tamoxifen, an agent that will induce their cancer-causing gene, and then treat them with either UVA, UVB, or no UV at all. After their treatments I will be able to monitor them for tumor development and collect data from this colony and them analyze as I did for the black mice.
This summer has helped me grow a lot and really learn how to problem solve. I have become really invested in my project and I am very excited for the next steps to come. I feel as though I have become a lot more independent and that I am being given more tasks to complete on my own. It has been difficult to maintain my confidence when experiments fail, especially when there are repeated failures, but I don’t stay discourage for long. With help from my mentors and peers in the lab I have been able to work through and around my problems to find solutions. I am very glad that I had this experience and I am very excited to see the outcome of my project in the future.