The role of Fat1b protocadherin proteins in hair-cell regeneration

My project this summer involved injecting a translation-blocking morpholino oligonucleotide into transgenic zebrafish embryos to knock down the function of a gene called fat1b. We hypothesize that fat1b may limit hair-cell progenitor proliferation and hair-cell regeneration. After injecting the morpholino into the embryos, we waited until three days post-fertilization to image the hair cells. We noticed that some injected larvae had a bit more immature hair cells than uninjected larvae, so we decided to image larvae at four days post fertilization. Imaging at four days seems to be promising for demonstrating a difference in the number of hair cells in the absence of fat1b.

In order to determine if the morpholino was functioning, I started a cloning project. In this cloning project, a plasmid was designed to incorporate part of fat1b to which the morpholino binds, resistance to kanamycin, and a promoter that constitutively drives green fluorescent protein (GFP). We then transformed Escherichia coli with the plasmid. Out of the eight colonies that were chosen, about half had successfully taken up plasmid of interest. We chose two out of the half to extract DNA, which we hope to inject into embryos soon.

The highlight of my project so far is the cloning portion. Even though I am not done with that step, I was successfully able to create a plasmid by digesting restriction enzymes to the piece of fat1b we wanted. I found it fascinating that Dr. Steiner was able to design this type of plasmid by using an application on his computer. I was able to use my microbiology techniques in making the selective media to grow the transformed E. coli.

I’m excited to continue my research with Dr. Steiner in BIO 480: Research in Biology this upcoming semester. I will be looking forward to learning how to inject the extracted DNA and morpholino into one-cell stage embryos.

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