Date of Award
Doctor of Philosophy (PhD)
Richard K. Cooper
The goal of this work was to evaluate the feasibility of using antimicrobial peptide gene transfer to enhance oyster immunity. The specific objectives were to: (1) compare the efficiency of collagenase, pronase, and trypsin to dissociate atrium, ventricle and mantle into individual cells for the initiation of primary cell culture; (2) develop cryopreservation conditions to preserve the dissociated cells; (3) establish a serum-free transfection system; (4) evaluate inducible promoter function using the established serum-free transfection system; (5) evaluate the effects of the transfer of antimicrobial peptide genes on oyster immunity. Pronase was found to be the most effective enzyme for dissociating the atrium, ventricle, and mantle. Thereafter, pronase was used to dissociate atrium and ventricle tissues for development of cryopreservation conditions to preserve cells for culture. Dimethyl sulfoxide, glycerol, and propylene glycol were evaluated for their effectiveness as cryoprotectants. Freezing rates and thawing temperatures were compared to minimize the damage. The optimal conditions were 10% glycerol, freezing at a medium rate (vials containing cells were packed into a 21 x 19 x 7 cm and 0.7 cm thick polystyrene foam box and with 80 g of cotton followed by equilibration at 25°C for 20 min, -80°C for 16 h, and -196°C for storage) and thawing at 45°C for atrial cells, and 10% glycerol, freezing at a medium rate and thawing at 25°C for ventricle cells. A serum-free transfection system for ventricle cells was established. A heat inducible promoter was evaluated in the system. The optimal heat shock conditions for the heat shock protein 70 promoter from snail, Biomphalaria glabrala, was 40°C for 1 h followed by recovery at 25°C for 12 h. The induction of the cecropin B promoter from moth, Hyalophora cecropia, was evaluated in ventricle cells using lipopolysaccharide and IL-1-alpha. In vivo transfer of an antimicrobial peptide gene was conducted. Although not significantly different, oysters receiving the antimicrobial genes controlled by the cecropin B promoter tended to have lower bacteria tissue loading than the controls. The results of this work implied that antimicrobial peptide gene transfer to enhance oyster immunity is feasible.
Cheng, Ta philip Chih, "Evaluation of Heterologous Promoter Function in the Eastern Oyster (Crassostrea Virginica)." (2000). LSU Historical Dissertations and Theses. 7250.