TWENTY-THIRD ANNUAL EASTERN FISH HEALTH WORKSHOP JOHN CARVER INN, PLYMOUTH, MA 30 MARCH - 2 APRIL, 1998 Expression, In Channel Catfish Ovary Cells, Of A Red-Shifted Green Fluorescent Protein Genetic Variant With Human Codon Usage Mutations Steven E. Poet and Victoria V. Burnley University of Georgia, College of Veterinary Medicine, Department of Medical Microbiology and Parasitology, Athens, GA 30602-7386 Green fluorescent protein (GFP) is a novel genetic reporter system for expression of heterologous gene sequences. This reporter protein has the advantage of being detected directly from living tissues instead of fixed samples. When excited with specific wavelength light, GFP emits bright green fluorescence. Red-shifted GFP variants have been developed that can be excited with more conventional fluorescence filter sets. These variants can emit green light up to 35 times more intense than wt GFP variants. Unfortunately, the red-shifted GFP variants (EGFP) have been designed for mammalian expression by incorporating human codon optimized silent base changes. These silent mutations in the EGFP gene enhance expression, and fluorescence, in mammalian and plant cells, but inhibit expression in other organisms, such as yeast. Data from codon usage tables for human, mouse, catfish, and yeast suggest that EGFP should be efficiently expressed in channel catfish cells. In order to test the hypothesis that EGFP will be expressed in channel catfish ovary cells, the eukaryotic expression plasmid pEGFP-C1 was transfected into channel catfish ovary cells. Fluorescence was monitored using conventional FITC fluorescence microscopy with excitation at 488 nm. Bright green fluorescence was observed in pEGFP-C1 transfected cells, but not in mock transfected, or irrelevant gene transfected cells. These data suggest that EGFP can be used as a reporter gene for future DNA-mediated vaccination experiments in channel catfish.   Return to 23rd Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page