Date of Graduation

5-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Huang, Yan

Committee Member

Nelcon, Christopher E.

Second Committee Member

Kwon, Young Min

Third Committee Member

Nakanishi, Nagayasu

Keywords

CRSIPR-Cas9; ELOVL2 integration; Growth performance; LC-PUFAs; Lipid accumulation; Zebrafish

Abstract

Fish are a rich source of EPA and DHA, which are well-known long-chain polyunsaturated fatty acids (LC-PUFAs). The sustainable production of these essential fatty acids poses significant challenges, mainly due to our dependence on marine fish and certain nuts. CRISPR-Cas system was utilized to insert the elongation of long-chain fatty acids-like2 (Elovl2) gene from Salmo salar into Danio rerio. Elovl2 was introduced at 10-15-minute post-fertilization, and successful integration confirmed by PCR genotyping, sequencing and expression of EGFP. Approximately 67% of injected embryos successfully exhibited EGFP and sequence results verified the gene integration of Salmo salar Elovl2 at the first start codon. Although Injected embryos exhibited lower hatching rates and higher mortality compared to the wild-type groups, but they successfully expressed the inserted Elovl2 gene, showing approximately a 7-fold increase at the early stages.

GC-MS and RT-qPCR analysis of fatty acid profiles showed that fatty acid biosynthesis-related gene (Elovl2, Fads2, Cpt1a) and adipogenesis-related genes (Fabp4, Pparg, Cebpa) were significantly upregulated in transgenic groups, with significant increases in omega-3 fatty acid levels across various body regions, including the head, trunk and tail at 30-, 60-, and 90-day post fertilization after fed different diets (brine shrimp or commercial diets). Moreover, length (mm), weight (mg) and depth (mm) were increased significantly in transgenic groups compared to the control groups, with further confirming of the RT-qPCR analysis showing the significant upregulation of growth-related genes (Ghr-a, Ghr-b, and Igf1a) and myogenesis-related genes (Myf5, Myf6, Myog, and Myod1) in the trunk and tail regions. Histological analysis also verified the increases of muscle fiber diameter in transgenic groups fed different diets, establishing a strong correlation between gene expressions and muscle hypertrophy. These findings suggest direct evidence of combining genome editing with ideal feeding plans that can significantly enhance aquaculture performance.

Overall, our findings demonstrate that incorporating the Salmo salar Elovl2 gene into zebrafish (freshwater species) enhances omega-3 fatty acid production and improves growth performance. It could be a viable approach to enhance the aquaculture productivity of species with low omega-3 content, providing a sustainable solution for improving fish quality and meeting the growing demand for nutritious aquaculture products.

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