The glutathione (GSH) pathway is an antioxidant pathway found in humans and zebrafish. This GSH pathway plays a critical role in detoxifying cells from substances such as methylmercury (MeHg), which is a prevalent environmental contaminant in the Great Lakes region. Genetic variations in certain GSH genes have been reported to affect methylmercury detoxification efficiency in adult tissue; however, less is known about these environment-gene interactions during embryonic development. To investigate this context, we are creating knockout zebrafish lines with loss-of-function mutations in the GSH pathway genes gclm and gstp2. Mutations were created by microinjection of CRISPR reagents into 1-cell zebrafish embryos. Offspring of mature zebrafish identified with potential mutation were genotyped to characterize the DNA sequence at the CRISPR target area using PCR + restriction enzyme digestion with gel electrophoresis. The purpose of this project is to confirm loss-of-function mutations and generate homozygous mutant zebrafish lines to study how these genes influence developmental toxicity of contaminants during development.
