I believe as time has passed, I’ve made some good progress, especially since my last blog post. I used to struggle the most with injecting my fish embryos with morpholino oglionucleotide, as well as using the confocal microscope. However, now I find both techniques easy and rather fun. This was a big challenge I overcame and now feel confident doing. By being able to do both these crucial steps in my study, I’ve started to collect some very interesting data. I’m seeing that at some specific concentration of morpholino, under the confocal microscope there seems to be an increase in the amount of hair cells within each neuroblast. This was very exciting for both my mentor and I.
Finding the concentration of the morpholino to inject was rather hard. Often times my injections were killing the embryos because the concentrations were too high. Many trials needed to be done to find the right amount to inject. One of the biggest things I’ve learned through this experiment was to be patient. I never expected it to take so much time. However, it was my patience and practice that finally allowed me to get good and promising results. My next step in my study is to kill off the hair cells in my fish by use of copper treatment in order to measure the rate of growth and cell number of the hair cells.
Since the last time I posted a blog, I have conducted my experiment about 4 or 5 times. Each time, I have improved on my techniques. The hardest technique that I have yet to perfect would be the injection of morpholino oglionucleotides into the zebrafish embryos. This technique is very difficult and is the basis of my experiment. As far as extracting RNA, making cDNA, running PCR and gel electrophoresis, I believe i have those down pact at this point. I have done them enough times to be confident in my abilities.
Because I can’t get data without injecting properly, I haven’t retrieved as much data as I would have liked. However, from the data that I have collected, I found that the morpholino oligonucleotide that I have been trying to inject, has been knocking down the gene I’ve been trying to knock down. My plan is to work throughout winter break to continue practice of injecting the fish embryos. I am confident I will be able to do it properly soon. After I can properly inject my fish embryos, my next step is to kill the hair cells by copper treatment to measure the rate of growth and cell number of the hair cells.
The title of my project is “Fat1 Protein and Hair Cell Regeneration”. The purpose of my research is to look at the role Fat1B has on regenerating hair cells. Often times hearing loss is due to the loss of sensory hair cells in our ear. These hair cells convert sound waves into messages. In humans, hair cells don’t regenerate after they die, however in zebrafish, hair cells are found in the lateral line, a sensory system that detects water movements, but do regenerate after being damaged or destroyed.
My project essentially is to understand how the hair cells in zebrafish regrow. Working under Dr. Steiner, I know I will learn a lot about the process of regenerating hair cells in zebrafish and how we might be able to use this process on humans when fully understood. I have already learned many techniques and protocols in the lab, and I know that everyday I will continue to grow as a researcher and gain more knowledge in the fields of molecular biology and bioinformatics.
I’m looking to see if a gene called Fat1B controls cell proliferation and regeneration of the sensory cells in the lateral line of these fish. To look at the role of Fat1B, I have to knockdown the gene’s expression. I do this by injecting morpholino oligonucleotide, which bind to certain spots in their pre-mRNA preventing normal splicing and making normal proteins, into zebrafish embryos. My project looks at different concentrations of morpholino used to see each effect on hair cell regeneration. Afterwards, I will extract RNA from both the injected and uninjected embryos and use a method called PCR to see if the morpholino I injected is correctly working. Aside from the bigger methods I just described, I have also learned many small yet important techniques such as calibrating injecting needles and using both the florescent and confocal microscope. Every technique learned, whether big or small, are important because they can potentially contribute to future therapies for hearing loss.