In vivo transfection of adult eastern oysters Crassostrea virginica

The eastern oyster Crassostrea virginica provides a commercially valuable i ndustry along the eastern and Gulf coasts of the United States. Recently th is industry has been damaged by disease problems, creating an interest in t he use of gene transfer (transfection) to improve disease resistance. We tr ansfected adult oysters with two genes, red-shifted green fluorescent prote in (rsGFP), commonly used as a reporter gene, and the lytic peptide cecropi n B (cepB), known to have antimicrobial properties. Oysters were transfecte d by injecting DNA mixed with SuperFect (TM), reagent (Qiagen Inc.) into th e adductor muscle sinus. Oysters were assigned to three groups of 15: the f irst was injected with rsGFP complexed with transfecting reagent; the secon d 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 r eaction (PCR), and hemocytes were examined by flow cytometry and fluorescen ce 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 da y 4, and in 15 of 15 oysters at day 10. The cepB gene was detected by PCR i n 12 of 15 oysters at day 4 and in 14 of 15 oysters at day 10. No oysters f rom the control group were positive for either gene at days 4 or 10. Green fluorescence was detected by How cytometry at significantly higher levels ( P < 0.05) in oysters injected with rsGFP than in other oysters at day 4, bu t not at day 10. This report indicates the ability to introduce DNA into ad ult eastern oysters with subsequent gene expression. Future work will invol ve developing these techniques for enhanced disease resistance in oysters.