Date of Project

5-2-2023

Document Type

Honors Thesis

School Name

College of Arts and Sciences

Department

Biology

Major Advisor

Dr. Mary Kroetz

Second Advisor

Dr. Josef Jareczek

Abstract

Despite its widespread use in research, the model organism C. elegans has several biological processes like gonadal development with potentially unexplored genetic regulators. Previous transcriptome analysis has identified several genes that are upregulated in a specific tissue or sex during the development of the somatic gonad in C. elegans (Kroetz et al. 2015) that have not been previously connected to this process. Of these genes, this research is concerned with chk-1 and fasn-1. Abrogating the expression of these genes in gonadal tissue during gonadogenesis could cause a change in phenotype for affected C. elegans that would aid in understanding these genes’ regulatory role in this process. To accomplish this, we use CRISPR-Cas9 and homology-directed repair to modify the genome of C. elegans so that the proteins chk-1 and fasn-1 produce are tagged with GFP. These tagged proteins can be selectively degraded by the transgene GFP-nanobody-ZIF-1 degron system in somatic gonad tissues to achieve the desired abrogated expression of chk-1 and fasn-1 (Wang et al. 2017). To modify these genes with CRISPR, gRNA, and homology directed repair constructs were designed and created for each gene. With provided Cas9 endonuclease, the gRNA and homology directed repair construct would be injected into the germ cells of individual C. elegans to modify the genes-of-interest so that they produce GFP-tagged proteins. Future research would include cross-breeding the C. elegans animals containing GFP-tagged proteins with those containing the degron system to generate an animal containing both the GFP-tagged genes and the degron system in their genome.

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