Development and validation of gene delivery methods for Crassostrea virginica

The Eastern oyster, (Crassostrea virginica), an important part of coastal economies because aquaculture creates jobs as well as attract tourists. Sadly, the oysters are under constant threat due to increasing pollution, red tides, and diseases. Bivalves and oysters in particular are also becoming potential model organisms in medical research. With the oyster genome sequenced, scientists are focusing on deciphering the function of the predicted genes. However, the limited number of molecular and cellular tools available makes decoding the genome challenging. A consistent gene delivery system needs to be developed to interrogate the genome and understand the oyster phenome. In this study, we adapted and furthered early methodologies for delivering DNA into oysters, we used both chemical (dendrimers) and physical (electroporation) gene delivery into oyster hemocytes in vivo and in vitro using plasmids under CMV (pmax-GFP) and SV40 (pTOPO-GFP) promoter control with green fluorescent protein (GFP) tagged genes. We observed GFP expression as early as 24 hours post transfection in both in vivo and in vitro studies using confocal microscopy. Indirect immunofluorescence analysis co-localized GFP with the anti-hemagglutinin antibody in C. virginica hemocytes expressing C. gigas vascular endothelial growth factor fused with hemagglutinin and GFP (pCgVEGF-HA-GFP). In addition, flowcytometry analysis evaluated as 0.5% GFP expressing in vivo and 0.03% in vitro GFP expressing hemocytes. Here, we provide the basic methodology to understand the gene functions and mechanisms that underlie oyster physiology and ecology, in order to develop oyster cell lines.