Date of Project

4-28-2023

Document Type

Honors Thesis

School Name

College of Arts and Sciences

Department

Biology

Major Advisor

Mary Kroetz

Second Advisor

Paul Kiser

Abstract

The gene pros-1 is a transcription factor that is highly expressed within neuronal sheath cells, glial cells, and excretory canal cells. pros-1 plays a role in cell determination of those cell types in the nematode C. elegans, which promotes organismal development. But the degree to which pros-1 presence is important is still not fully understood, because there are many genes involved in development that when mutated or damaged can result in unexpected phenotypes or even total loss of function to a certain developmental mechanism. What makes pros-1 valuable to research is that it is a functional homologue to a gene found in humans called prox-1, a transcription factor that is vital in the proper neurogenesis of the central nervous system in the human body and altered levels of the PROX-1 protein have been linked to several forms of cancer. Recent data has shown that pros-1 is highly expressed in the somatic gonads of male C. elegans.

We want to understand whether the pros-1 genes plays a role in the development of the somatic gonad, however this is complicated by the fact that pros-1 expression in the excretory canal cell and nervous system is required for the survival of the animal. Our project has developed a system of gonad-specific protein silencing to investigate the essential factor of target genes on the development of the somatic gonad in C. elegans. This is achieved by crossing genetically altered nematode strains to generate progeny of C. elegans that contain a protein silencing tool that allows for the degradation of a green fluorescent protein (GFP) tag that is attached onto pros-1.

Degradation is performed by a tissue specific targeting system called a degron system that utilizes the proteosome. Degrons utilize small nanobodies to target a fluorescent protein fused to the target protein and then bring the fluorescently tagged protein to the proteosome, silencing the expression of our target gene pros-1. The degron system is only expressed in the gonad to target the pros-1-GFP fusion protein. This system will allow for the removal of the essential gene pros-1 in the male gonads, and to determine the loss of function phenotype.

GFP is a versatile biological marker because its protein structure fluoresces a bright green light when excited by a certain light emission, and is commonly used to visualize protein localization, and detecting transgenic expression in vivo. We will be able to confirm the effectiveness of this protocol by the amount of GFP fluorescence in degraded vs nondegraded strains of C. elegans as well as by determining if the male gonad is misinformed during development when pros-1 is removed. Once the C. elegans have the pros-1::GFP fusion and the ckb-1::degron fusion, PROS-1 will fluoresce in all locations it is expressed in except the somatic gonad because that is the only location the degron construct is also being expressed and thus binding to the GFP tag and subjugating the protein to degradation. This overall expression system provides an efficient and testable system for tissue specific protein silencing in within C. elegans which will work to understand the role of pros-1 in the function of the male somatic gonad.

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