Transfection of Eastern Oyster (Crassotrea virginica) Embryos

Abstract

There is a need for research in disease resistance and microbial elimination 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 resistance to neomycin and related antibiotics such as G418. The antibiotic G418 was 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 duration decreased survival (P <.05). Chemically mediated transfection did not significantly affect survival (P =.5172). Transgenic larvae were identified 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. Significant differences in survival between transfected and nontransfected larvae were detected for electroporation (P =.0147) and chemically mediated transfection (P =.037). Gene transfer was also confirmed with polymerase chain reaction and observation of expression of green fluorescent protein. This study documents the first successful insertion and expression of foreign DNA in eastern oyster larvae.

Disease and mollusc quality

“…Technologies using gene transfer to embryos and eggs have also shown promise (Buchanan et al, 2001;Cadoret, 1992;Cadoret et al, 1997a,b;Mialhe et al, 1997;Powers et al, 1995) but need to be improved to become cost effective and produce higher survival rates in recipient animals before commercialisation can be considered feasible.…”

Effects of air exposure on the lysosomal membrane stability of haemocytes in blacklip abalone, Haliotis rubra (Leach)

“…The neutral red stock solution consisted of 2.28 mg of neutral red powder dissolved in 1 mL of dimethyl sulphoxide (DMSO). The working solution (0.01 mg mL −1 ) was prepared by diluting 17 μL of the stock solution with 4 mL of artificial saline solution consisting of 0.48 g L −1 CaCl 2 , 1.45 g L −1 MgSO 4 , 2.18 g L −1 MgCl 2 ·6H 2 O, 0.31 g L −1 KCl, 11.61 g L −1 NaCl and 0.35 g L −1 NaHCO 3 (Buchanan, Nickens, Cooper & Tiersch 2001).…”

The effect of size on the response of Pacific oysters (Crassostrea gigas) to changes in water temperature and air exposure

“…The working solution (0.04 mg ml À1 ) was prepared by diluting 17 ml of the stock solution with 1 ml of oyster saline solution (Buchanan et al 2001).…”