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.
This summer Dr. Steiner and I will be continuing our research on the role fat1b may have on the proliferation and regeneration of sensory cells in the lateral line sensory organs of the zebrafish. Fatlb is an ortholog of the fat1 gene, which encodes a protein that limits progenitor-cell proliferation in a variety of model organisms. The sensory hair cells found in the lateral line along the side of the fish are similar to mammalian hair cells. However, the stark difference is that the zebrafish hair cells are capable of regenerating after damage. By understanding the pathways that govern the proliferation and regeneration in this model organism, therapies may be developed to treat hearing loss in humans.
We will be using a different morpholino oligonucleotide sequence to knock down the function of Fatlb. Last summer we used a morpholino that targets a splice site in the pre-mRNA, however this year, we will be using a morpholino that targets the translation start site of Fatlb. This inhibits the mRNA from entering into the ribosome for protein synthesis.
We will continue injecting the translational morpholino into embryos and analyzing the results. In order to make sure that the designed morpholino is working, we will be conducting a cloning project to ensure that Fat1b is knocked down. In this project, we will make a plasmid that incorporates the part of fat1b to which the morpolino binds, resistance to kanamycin, and a promoter that constitutively drives green fluorescent protein (GFP). We will then transform Escherichia coli with the plasmid and pick colonies that have successfully taken up the plasmid of interest. DNA will be extracted from the selected E. coli and injected into embryos. Injected fish should be green under fluorescence, whereas fish injected with the plasmid and fat1b translational morpholino should be devoid of GFP.
My goal for this summer is to make much more progress than I have in the past. Unfortunately last year we did not make as much progress as we would have liked, but this year’s new approach looks promising. I’m also excited to further enhance my knowledge and skills in a laboratory setting. I recently learned how to dechorionate larvae and I will learn how to precisely inject into a zygote for my cloning project.