Gene Editing in Zebrafish for Modeling Nicotine Dependence
Three genes for nicotinic acetylcholine receptors, the alpha 3 (chrna3), the alpha 5 (chrna5) and the beta 4 (chrnb4) gene have been linked to heavy smoking and smoking onset in humans. Mutations of these genes could increase the risk for developing lifelong nicotine dependence. To explore these potential risk factors we use zebrafish and apply molecular tools including CRISPR/Cas9 for generating gene-knockout mutations and CRISPR/BE4max for generating gene-editing mutations. These tools will allow us to determine if the chrna3, chrna5 and chrnb4 gene mutations are associated with changed nicotine sensitivity, nicotine seeking or nicotine avoidance behavior in zebrafish. Overall, one specific goal of the project is to develop zebrafish lines (or strains) without functional chrna3, chrna5 and chrnb4 genes (gene-knockout mutants). We successfully identified potential gene-knockout founder fish (F0) generated with the CRISPR/Cas9 method for the chrna3, chrna5 and chrnb4 genes. In addition, we were able to generate the first homozygous and heterozygous chrna3 gene knockout mutants and the first heterozygous chrna5 gene knockout mutants that we obtained through the Zebrafish Sanger Mutation project. In summary, we made significant progress towards establishing stable zebrafish homozygous and heterozygous gene knockout mutant lines for three nicotinic acetylcholine receptor genes that will be used for behavioral testing of zebrafish nicotine-seeking and avoidance behavior.
Porter, Sophia; Ngoc, Thanh; Nguyen, Thien; and Fagan, Avery, "Gene Editing in Zebrafish for Modeling Nicotine Dependence" (2023). Annual Student Research Poster Session. 124, Scholarly and Creative Work from DePauw University.