Title

A study on the production of transgenic medaka, Oryzias latipes, resistant to bacterial infection by introducing a cecropin gene

Date of Completion

January 2000

Keywords

Biology, Molecular|Biology, Cell|Agriculture, Fisheries and Aquaculture

Degree

Ph.D.

Abstract

In this study whether the expression of cecropin antimicrobial peptides affects the resistance of transgenic medaka to pathogenic bacteria was investigated. Prior to the production of transgenic fish, a first set of experiments was conducted to test whether bactericidally active cecropins could be expressed in a fish cell line. Chinook salmon embryo cells (CHSE-214) were transfected with cecropin transgene constructs; Hyalophora cecropia preprocecropin B, procecropin B, cecropin B and porcine P1 cecropin. From the transfected cells, single cell clones were selected and screened for the presence of cecropin gene constructs by PCR amplification. The expression of the cecropin transgene in the PCR positive clones was determined by RT-PCR. Southern blot hybridization results showed that the cecropin gene constructs were integrated into the genome in a multiple integration pattern. Bactericidal activity of the cecropins synthesized from the transgene constructs was detected using inhibition zone assay for fish pathogenic bacteria; Aeromonas hydrophila, Pseudomonas fluorescens, and Vibrio anguillarum. Cecropins produced in CHSE-214 cells possessed bactericidal activity against these three bacteria. ^ Upon observation of expression of cecropins in CHSE-214 cells, a second set of experiments was undertaken to determine expression of cecropin in transgenic fish. The gene constructs described above were electroporated into newly fertilized medaka eggs. Transgenic fish were identified by PCR amplification of the transgene sequence. Then P1 transgenic individuals were crossed with nontransgenic fish to obtain F1 progeny. F1 transgenic fish were crossed with nontransgenic control fish to produce F2 transgenic fish. The expression of the transgene was determined using samples from the F 2 transgenics by RT-PCR assay. Southern blot hybridization results indicated that cecropin transgenes were integrated into the F2 transgenic fish genome. To determine the resistance of transgenic fish to fish pathogenic bacteria, nontransgenic control fish and F2 transgenic fish were challenged with P. fluorescens and V. anguillarum . The results from the challenge experiments demonstrated that transfer of a cecropin gene can enhance resistance of transgenic fish to fish pathogen bacteria. ^