Date of Award

1999

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Oceanography and Coastal Sciences

First Advisor

Charles A. Wilson

Abstract

The eastern oyster, Crassostrea virginica, supports a valuable industry in the United States and Louisiana. In recent years this industry has been plagued by diseases, creating interest in the use of gene transfer to produce oysters capable of eliminating pathogens. The goal of this work was to develop techniques for laboratory research of eastern oysters, and for transfer of genes to oyster gametes, larvae, and adults. Cell culture provides an effective alternative to work in whole animals. Cell attachment and spreading on various culture substrates was improved, and poly-D-lysine was identified as a superior substrate. Toxicity of the antibiotic G418 to oyster cells was determined for use as a selective agent, and techniques for gene transfer in cells were identified. Adult oysters were conditioned for spawning in a recirculating system, although declines in physiological condition were noted. Electroporation of oyster sperm did not affect larval production, but did allow transfer of the gene for red-shifted green fluorescent protein (rsGFP). Larvae expressing rsGFP were observed with fluorescent microscopy and presence of the gene was verified by the polymerase chain reaction (PCR). Transgenic oyster larvae were also produced by electroporation and chemically mediated transfection (with SuperFect(TM); Qiagen Inc.) of embryos. Transfer of the aminoglycoside phosphotransferase II gene (neo ') by both methods resulted in increased larval survival by conferring resistance to G418. Larvae expressing rsGFP were observed with fluorescent microscopy, and presence of the gene was verified by PCR. Transgenic oyster adults were produced by injection of rsGFP or the cecropin B gene complexed with SuperFect(TM) into the adductor muscle sinus. Presence of the gene in DNA of treated oysters was verified by PCR after 4 d (87%) and 10 d (96%). Significant differences in green fluorescence were detected by flow cytometry in hemocytes after 4 d, but not after 10 d, and hemocytes expressing rsGFP were observed with fluorescent microscopy. These studies improved culture conditions and gene transfer in oyster cells, improved maintenance of oysters in the laboratory, and documented gene transfer and gene expression in oyster gametes, larvae, and adults. This is the first report of gene transfer in the eastern oyster.

ISBN

9780599635951

Pages

256

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