Date of Project

4-29-2023

Document Type

Honors Thesis

School Name

College of Arts and Sciences

Department

Biology

Major Advisor

Dr. Mary Kroetz

Second Advisor

Dr. Paul Kiser

Abstract

In C. elegans, the gene glp-1 encodes for a Notch receptor called GLP-1, one of two found in C. elegans’ genome. The gene has been previously implicated in the development of the hermaphroditic germline as well as playing a role in the mitosis/meiosis decision. Genetic screening has further identified it as potentially playing a role in the development of the male somatic gonad, making it an ideal candidate for a reverse genetic. We did this by silencing glp-1 and observing if any alterations to the gonad’s phenotype occur.

Normally this could be done by performing a gene knockout. However, due to the nature of Notch receptors’ overall role in the regulation of tissue development, it is an essential gene, and silencing it would result in worms dying as embryos, long before a gonad capable of being studied could form. In order to conduct a knockdown study, CRISPR-Cas was used to tag GLP-1 at its C-terminus with green fluorescent protein (GFP). In a separate strain of worms, degron, a method used for targeting proteins tagged with GFP for ubiquination and lysis, was placed under a promoter specific to the gonad. When the two strains are crossed, they will result in a worm that has functional GLP-1 in all tissues except for the gonad.

In order to resolve the sex ratio issue and focus on the development of the male gonad, which has been neglected in the literature, a mutant strain of him-8 worms that produced offspring with a ratio of three males for every ten viable was cultivated. The bulk of our work was spent crossing the degron strain with the him-8 strain, resulting in a new strain of worms that express degron in the gonad and are disproportionately likely to be male. By coupling this strain with the use of CRISPR to tag any gene of interest with GFP, a gonad-specific knockdown study can be performed not only for glp-1 but for any gene expressed in the gonad, essential or not.

We crossed the new strain with degron and the mutant him-8 with the glp-1::GFP worms, generating both a produce a worm homozygous for all three genes, which should thus contain adult male worms without GLP-1 in their gonads and a strain with him-8 and glp-1::GFP but not degron, allowing us easy access to a source of a male control group. These we will examine for alterations against the N2 male gonad. In doing so, we will hopefully further understand Notch receptors’ role both in the development of the male gonad as well as in general tissue development.

Download

Share

COinS