In Vivo transfection of adult eastern oysters Crassostrea virginica

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The eastern oyster Crassostrea virginica provides a commercially valuable industry along the eastern and Gulf coasts of the United States. Recently this industry has been damaged by disease problems, creating an interest in the use of gene transfer (transfection) to improve disease resistance. We transfected adult oysters with two genes, red-shifted green flourescent protein (rsGFP), commonly used as a reporter gene, and the lytic peptide cecropin B (cepB), known to have antimicrobial properties. Oysters were transfected by injecting DNA mixed with SuperFectTM reagent (Qiagen Inc.) into the adductor muscle sinus. Oysters were assigned to three groups of 15: the first was injected with rsGFP complexed with transfecting reagent; the second was injected with cepB complexed with transfecting reagent; and the third was injected with saline (control group). Hemolymph was collected at 4 and 10 d after injection. DNA was extracted for analysis by polymerase chain reaction (PCR), and hemocytes were examined by flow cytometry and flourescence microscopy for detection of green flourescence due to rsGFP expression. The rsGFP gene was detected by PCR in hemocytes from 14 of 15 oysters at day 4, and in 15 of 15 oysters at day 10. The cepB gene was detected by PCR in 12 of 15 oysters at day 4 and in 14 of 15 oysters at day 10. No oysters from the control group were positive for either gene at days 4 or 10. Green flourescence was detected by flow cytometry at significantly higher levels (P < 0.05) in oysters injected with rsGFP than in other oysters at day 4, but not at day 10. This report indicates the ability to introduce DNA into adult eastern oysters with subsequent gene expression. Future work will involve developing these techniques for enhanced disease resistance in oysters.

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Journal of the World Aquaculture Society

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