Transfection of eastern oyster (Crassotrea virginica) embryos

There is a need for research in disease resistance and microbial eliminatio n in the eastern oyster Crassosostrea virginica. Gene transfer may lead to advances in this area, and a means of selecting transfected larvae would be useful. We transfected 3-hour-postfertilization embryos with the bacterial gene aminoglycoside phosphotransferase II (neo(r)), which confers resistan ce to neomycin and related antibiotics such as G418. The antibiotic G418 wa s examined as a potential selective agent. A neutral red assay was used to determine survival after 48 hours of exposure to various concentrations of G418 (0-4 mg/ml). We examined the effects of electroporation and chemically mediated transfection of 3-hour- postfertilization embryos on survival to straight-hinge larvae. DNA alone was found to have no effect on survival (P > .05). For electroporation we found that increased voltage and pulse dura tion decreased survival (P < .05). Chemically mediated transfection did not significantly affect survival (P = .5172). Transgenic larvae were identifi ed after electroporation and chemically mediated transfection. These larvae were reared for 24 hours and exposed to G418 at 0.3 mg/ml for 48 hours. Si gnificant differences in survival between transfected and nontransfected la rvae were detected for electroporation (P = .0147) and chemically mediated transfection (P = .037). Gene transfer was also confirmed with polymerase c hain reaction and observation of expression of green fluorescent protein. T his Study documents the first successful insertion and expression of foreig n DNA in eastern oyster larvae.