Meet Neeraja Balachandar
Major: Neuroscience, Public Health minor
Graduation Year: May 2017 Field of Study: Developmental Neuroscience Abstract: PTF1A is a bHLH transcription activator that specifies neural progenitor cells in the dP4 domain of the developing neural tube to the inhibitory fate. PRDM13 is a transcription factor expressed in the dP2-6 domains of the developing neural tube and found downstream of PTF1A in the dP4 domain. PRDM13 is a member of the PRDM family of proteins, many of which are involved in gene regulation. It is made up of an N-terminus PR domain and four zinc fingers,which are the functional portions of the protein, at the C-terminus. The PR domain is 20-30% identical to the SET domain present in histone lysine methyltransferases, which function in transcriptional silencing. PRDM13 plays a key role in specifying neural progenitor cells to an inhibitory fate by repressing genes coding for the excitatory fate. However, the anti-PRDM13 antibody our lab currently has is not very effective, impeding our ability to further study the function of PRDM13. Therefore, our lab has created a new mouse line using the CRISPR/Cas9 system to incorporate a V5 epitope tag at the C-terminus of the PRDM13 protein. Characterization of this new PRDM13-V5 tagged mouse line is vital to ensure that the tag doesn’t affect the function or expression of the protein and that it can be detected by the anti-V5 antibody through immunohistochemistry. Immunohistochemistry data shows that the mice homozygous for the V5 tag phenocopy those that are heterozygous for the tag as well as wildtype mice in the expression of the transcription factors: PAX2, PTF1A, TLX1/3, and LHX1/5. Additionally, in light of data from our lab that showed that a mouse line with an early translational stop codon in the PR domain of the protein still produced viable mice, the presence of multiple isoforms of PRDM13 was tested. Our GFP-Prdm13-Fus mouse line (GFP located at the N-terminus of PRDM13) was crossed with our new Prdm13-V5 line producing embryos that were heterozygous for GFP at the N-terminus of the protein as well as for the presence of the V5 tag at the C-terminus. Co-localization between GFP and V5 was examined in cells of these neural tubes. V5 and GFP appeared to colozalize in all cells, however due to certain limitations of this experiment such as the possibility of multiple isoforms of the protein in the same cell, we cannot discount the existence of multiple isoforms of PRDM13. Further experimentation, in the form of western blots, must be done to determine with certainty whether or not multiple isoforms of PRDM13 exist. I made the most of my experience by attending seminars and talks, by asking questions, and reading scientific papers about my field of study
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My Experience:
The Green Fellowship was an eye opening and educational experience for me. I learned about the various aspects of being a successful scientist -- the importance of attention to detail, perseverance, collaboration, effective time management skills, and thinking critically and creatively. I made the most of my experience by attending seminars and talks given both by colleagues in the lab I was working in as well as other scientists, asking questions, and reading scientific papers about my field of study. Conducting research full time was a novel experience that presented itself with its own unique challenges, but I truly believe myself to be a better thinker, scientist, and student as a result of it. |
My Advice:
You will be given a lot of independence and responsibility in the lab. Don’t let this daunt you! Take advantage of this opportunity to accomplish and learn as much as you can. Make sure you invest an appropriate amount of time necessary in lab. Ask questions when you have them and remember there are no stupid questions in science. You will make mistakes but don’t let them discourage you. Make every effort you can to learn from your mistakes because those are the lessons that you will remember. Last but not least, make connections with your colleagues and enjoy your experience! |
Make every effort you can to learn from your mistakes because these are the lessons you will remember |