Week 5:
University of Iowa Department of Anatomy and Cell Biology

July 7, 2013

Week 5 has passed, and I’m well into Week 6. Time has flown by, and I’m starting to realize that I only have about three weeks left until I’m done with the FUTURE Program. Three weeks to finish up as much of my experiment as I can, make a poster, and be ready to present it. Three weeks hardly seems like enough time, yet I start another project. Who doesn’t love a challenge?

For about a week, I have been going through the process of cloning a gene called vmat2. In the neurons, there are vesicles that help the neurotransmitters travel to the membrane of the neuron to be released. Vmat2 is found in the membrane of these vesicles and affects the ability for neurotransmitters to go inside. Another member of the lab is cloning the pink1 gene that I have mentioned in previous  posts. We are looking to see one of these genes will rescue the touchtone zebrafish mutant. After the cloning process is complete, we will inject the gene first into wildtype zebrafish to make sure that nothing is toxic to the fish. We will then inject the mutant zebrafish and let them develop. At 48 hours, we will perform a behavioral test that just involves touching the embryos a bit to see if they move. For wildtype embryos, they will zip all around the dish when probed. Touchtone mutants, on the other hand, won’t move during this test. After 5 days, we will run another behavioral test which will be like the one I showed in a previous blog. The embryos will then either be used for insitu or qPCR. Basically, we will be looking to see if pink1 and TH2 (and maybe TH1) levels have improved.

With this project, I’ll be able to use previous results. I have data showing that pink1 expression in down in the touchtone mutants, and I also have data showing that when fish are placed under oxidative stress pink1 expression is elevated (wildtype embryos were placed in hydrogen peroxide). This just allows me to have data to compare my future results to which will allow me to interpret if there is improvement or oxidative stress.

With the nitrofuran experiment, Barbara and I are trying to get as much done as we can. We are continuing more trials to repeat the insitu procedure, qPCRs, and behavioral tests to make sure that our results are reproducible. We have also started taking pictures of our insitu results which gives us a basic idea if the expression of a certain gene has changed. So far, our results have shown that movement is being affected which suggests that the nitrofuran is affecting dopamine in these fish. In my last post, I mentioned the very large insitu that we were working on which was going to show us the amount of melanocytes in the fish. One of the things that we noticed was that the wildtype embryos in nitrofuran looked similar to the touchtone control embryos. Another thing that we noticed the embryos that were in both nitrofuran and PTU had more melanocytes compared to the embryos just in nitrofuran. This suggests that the PTU is preventing the melanocytes from being killed. It will be interesting what else we see as we continue!

For the FUTURE Program, there was a reception held at Dean Schwinn’s house, and she is the Dean of the Carver College of Medicine. Current and past participants of the FUTURE Program, hosts of the current participants, and a few other administrative members. It was a great experience because I was able to interact  with people in different departments. Even though I did feel a little out of place, I think it was good that I gained exposure to a new experience like this.

On an unrelated note, I was able to see my family on the 4th. We went out to my grandpa and grandma’s house where we met up with my mom’s two brothers and their families. It was great to see everyone and relax a bit!