This Is AuburnElectronic Theses and Dissertations

Gene Editing and Hormone Therapy to Control Reproduction in Channel Catfish, Ictalurus punctatus

Date

2019-04-23

Author

Qin, Guyu

Type of Degree

PhD Dissertation

Department

Fisheries and Allied Aquacultures

Restriction Status

EMBARGOED

Restriction Type

Full

Date Available

04-23-2021

Abstract

Channel catfish (Ictalurus punctatus) and their hybrids account for more than 60% of aquaculture production in the US. The catfish industry is being severely challenged during recent years as a result of higher operating costs, disease, and competition from cheaper imported frozen catfish. A potential future tool to help address this problem is the utilization of genetically engineered catfish. To sterilize channel catfish, transcription activator-like effector nuclease (TALEN) was applied to induce mutagenesis of catfish type gonadotropin-releasing hormone (cfGnRH) and follicle-stimulating hormone (FSH) genes through electroporation respectively. Zinc finger nuclease (ZFN) was performed to knock out the luteinizing hormone (LH) gene through electroporation. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was utilized to edit cfGnRH and LH genes through microinjection. Targeted cfGnRH and FSH gene mutagenesis were found in 52.9% and 63.2% P1 fish respectively through TALEN technology. Fish spawning, reproductive behavior and the hatching of their eggs were suppressed. Luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants, which were not significantly different from controls (P>0.05). Human chorionic gonadotropin (HCG) hormone therapy resulted in spawning rates of 80% for female mutants and 88.9% for male mutants, and mean hatch rate of 35.0% for F1 embryo, which was not significantly different from controls (P>0.05). ii LH gene mutagenesis was found around 60 bp upstream of the expected site in 38.9% P1 fish through ZFN technology. None of the female mutant fish could spawn without hormone therapy. P1 mutant males had lower spawning rate than controls with egg hatch rates of 1%. HCG hormone therapy resulted in good spawning and hatch rates for mutants, which were not significantly different from controls (P>0.05). No obvious effects on other economically important traits were observed after the knockout of reproductive genes in all P1 and F1 mutants using TALEN and ZFN technologies through double electroporation. CRISPR/Cas9 induced 50% mutagenesis at the targeted sites of cfGnRH and LH gene respectively. No obvious pleiotropic effects were observed in terms of body weight. CRISPR- Cas9 nucleases enabled higher efficient genome editing than ZFNs, similar efficient genome editing than TALENs, however with low hatchability through microinjection. Gene editing of channel catfish for reproductive confinement of gene-engineered, domestic and invasive fish to prevent gene flow into the natural environment appears promising.