In Vivo Transfection of Adult Eastern Oysters Crassostrea virginica |
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| Authors: | John T Buchanan Ta Chi Cheng Jerome F La Peyre Richard K Cooper Terrence R Tiersch |
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| Institution: | Department of Oceanography and Coastal Sciences, Louisiana State University, Baton Rouge, Louisiana 70803 USA;Department of Veterinary Science, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803 USA;Aquaculture Research Station, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803 USA |
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| Abstract: | 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 fluorescent 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 SuperFect? 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 transfectlng 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 fluorescence microscopy for detection of green fluorescence 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 fluorescence 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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