Date of Award

5-2015

Document Type

Honors Thesis

Degree Name

Bachelor of Science

Department

Biology

Advisor/Committee Chair

Jamie Belrose

Committee Member

Ben Szaro

Committee Member

Daniel Wulff

Abstract

Research into the regeneration of optic nerves in Xenopus laevis has determined that heterogeneous nuclear ribonucleoprotein K (hnRNP K) plays a crucial role in regulating the trafficking and translation of mRNAs essential for the organization of the axonal cytoskeleton. To further explore this role, our lab has turned to tools that can definitively elucidate hnRNP K’s translocation in-and-out of the nucleus, as well as directly quantitate its degradation rate, in vivo. An appropriate tool for such experiments is the monomeric Eos fluorescent protein (mEosFP), which can be stably and irreversibly photo-converted. This fluorescent protein naturally emits green light (~516nm) and upon photo-conversion with UV (~405nm), it stably emits red light (~581nm). Using traditional cloning techniques, we are fusing mEosFP (Xenopus codon specific) to Xenopus hnRNP K in a plasmid designed to generate 5’ capped mRNA through in vitro transcription. The resultant RNA will be microinjected into 2-cell stage Xenopus embryos, and these embryos will be allowed to mature to stage 22 (24 hrs. post-fertilization). From these animals we will create dissociated, embryonic spinal cord / myotome cultures. We will then photo-convert mEosFP using a Zeiss 710 confocal microscope and monitor the subcellular location as well as quantitate the resulting fluorescence. To achieve this, we will use time-lapse photography and ImageJ software, respectively. A series of controls will be included to ensure the incorporation of the fluorescent protein does not interfere with hnRNP K’s biological activity. This technique will enable us to track hnRNP K’s movements through the cell more accurately and quantify its turnover rate in vivo.

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