Date of Award

2020

Document Type

Honors Thesis (Open Access)

Department

Colby College. Biology Dept.

Advisor(s)

José A. Fernández Robledo

Second Advisor

David R. Angelini

Third Advisor

Ronald Peck

Abstract

The Eastern oyster (Crassostrea virginica) is an important part of the East Coastal USA economy because aquaculture creates jobs. 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 sequencing of the oyster genome, scientists are focusing on deciphering the function of the predicted genes. However, the limited number of molecular and cellular tools available makes functional annotation of the genome challenging. A consistent, replicable gene delivery system needs to be developed to assess gene function and understand the oyster phenome. In this study, we adapted and furthered early methodologies for delivering DNA into oysters. We tested both chemical (dendrimers) and physical (electroporation) gene delivery into oyster hemocytes, in vivo and in vitro, using plasmids under CMV (pm ax-GFP, pC gVEGF-HA-GFP) and SV40 (pMLS-SV40-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 antibodies in C. virginica hemocytes, expressing C. gigas vascular endothelial growth factor fused with hemagglutinin and GFP (pCgVEGF-HA-GFP). Transfection efficiency, using flow cytometry analysis, for in vitro and in vivo was 0.042% and 0.008% for pm ax-GFP, 0.061% and 0.01% for pMLS-GFP, and 0.071% and 0.009% for pCgVEGF-HA-IRES-GFP respectively. Here, we provide the basic methodology to understand the gene functions, and mechanisms that underlie oyster physiology and ecology, in order to annotate the oyster genome.

Keywords

Crassostrea virginica, Eastern oyster, Hemocyte, Gene, Gene Annotation, Transfection, Chemical Dendrimers, Electroporation

Download

Share

COinS