|
|
|
Abstracts
Workgroup: Genetics and Oyster Populations
Optimization of gene delivery for improved oyster health
Principal Investigator(s):
Richard K. Cooper, Department of Veterinary Science, Louisiana State University, rcooper@agctr.lsu.edu
Co-Investigator(s):
Terrence R. Tiersch, School of Wildlife and Fisheries, Louisiana State University; Jerome F. La Peyre, Department of Veterinary Science, La. State University
Funding Period: 10/1/99-9/30/01
Significant progress has been made in laying the ground work for the production of transgenic oysters. Many of the tools needed to insure the success of this project have either been completed, or are near completion. Gene delivery techniques have been developed using electroporation (625 v/cm, 25:F, and time constant of 0.3 msec were the optimal parameter tested) and Superfect(r) (1:3 ratio was optimal) to demonstrate gene transfer into oyster larvae. These techniques demonstrated gene delivery and expression, although expression in larvae was less than 1%. Low expression could be due a number of factors, but most likely is due to failure of the CMV promoter to work efficiently enough to express rsGFP above threshold levels. Detection of rsGFP expression was further complicated by a high degreee of auto-fluorescence in oyster cells that would mask low levels of expression. In an unrelated project, we have demonstrated that accumulation of GFP in cells will inhibit normal growth and lead to higher mortality rates when compared to control cells (Cooper, unpublished data) Due to the difficulties experienced in using rsGFP in oysters, we redesigned our vectors using luciferase under a combination of inducible and constitutive promoters to be used in the continuation of this work funded by GOIP.
To evaluate the potential for gene transfer and expression in adult oysters, two genes were tested: red-shifted green fluorescent protein (rsGFP) and cecropin B (cepB). When oysters were bled at 4 and 10 days post-transfection and PCR was conducted on DNA from oyster hemocytes, the number of animals positive for the genes being tested ranged from 80% to 100%. The results from this project demonstrate the feasibility of DNA delivery to adult animals and the preliminary data supports results obtained from transfected koi and channel catfish (Cooper and Enright, 1999).
IMPACTS AND/OR BENEFITS
The recent problems of the oyster industry associated with V. vulnificus have caused a reduced consumer confidence and a decreased demand for oysters. The result has been a decline in prices, which has affected all levels of the Gulf oyster industry. In addition, diseases affecting oysters have the potential for causing declines in available stocks. We are addressing these problems using biotechnology techniques that are becoming routine in other agricultural commodities. Optimum methods for producing and evaluating transgenic oysters are being developed. Specific questions can be answered about gene regulation in oysters and may lead to an all oyster construct or a lytic peptide controlled by an oyster promoter. This project, combined with research in sterilization of oysters may provide the oyster industry with a quality product to overcome negative public opinion and disease problems facing the industry.
PROJECT PUBLICATIONS:
Buchanan, J.T., T.R. Tiersch, and R.K. Cooper. 1999. Gene transfer in oysters. Louisiana Agriculture 42:13.
Buchanan, J.T., Cheng, T.C., La Peyre, J.F., Cooper, R.K. and Tiersch, T.R. 1999. in vivo transfection of adult oysters. Journal of Shellfish Research 18:324.
Buchanan, J.T., T. Cheng, J.F. La Peyre, R.K. Cooper, and T.R. Tiersch. 1999. Gene Therapy for Oysters. Louisiana Chapter Meeting of the American Fisheries Society, Baton Rouge, Louisiana, February 4-5. Book of Abstracts (Best Student Presentation).
Cheng, T., Buchanan, J.T., La Peyre, J.F., Tiersch, T.R. and Cooper, R.K. 1999. Optimization of reverse transcription polymerase chain reaction (RT-PCR) for use with the eastern oyster Crassostrea virginica. Journal of Shellfish Research 18:293.
Nickens, A.D., J.T. Buchanan, R.K. Cooper, T.R. Tiersch. 1999. Preliminary Examination of Gene Delivery in Larvae of the Eastern Oyster Crassostrea virginica. United States Chapter of the World Aquaculture Society Annual Meeting, Tampa Bay, Florida, January 27-30. Book of abstracts
Buchanan, J.T., A.D. Nickens, R.K. Cooper, and T.R. Tiersch. 2000. Gene Transfer to Eastern Oyster Embryos. United States Chapter of the World Aquaculture Society Annual Meeting, New Orleans, Louisiana, February 2-5. Book of Abstracts
Buchanan, J.T., C.G. Paniagua-Chavez, R.K. Cooper, and T.R. Tiersch. 2000. Research-scale Culture of Eastern Oysters. United States Chapter of the World Aquaculture Society Annual Meeting, New Orleans, Louisiana Annual Meeting, February 2-5. Book of Abstracts
Buchanan, J.T., T. Cheng, J.F. La Peyre, R.K. Cooper, and T.R. Tiersch. 2000. Transfection of Adult Eastern Oysters by Injection. United States Chapter of the World Aquaculture Society Annual Meeting, New Orleans, Louisiana, February 2-5. Book of Abstracts (Best Abstract Award).
Buchanan, J. T., A. D. Nickens, T. R. Tiersch, and R. K. Cooper. 2000. Transfection of Eastern Oyster Embryos. National Shellfisheries Association Annual Meeting, Seattle, Washington, March 19-23. Journal of Shellfish Research 19:613.
Buchanan, J.T., A.D. Nickens, R.K. Cooper, and T.R. Tiersch. 2000. Techniques for transfection of eastern oyster (Crassostrea virginica) embryos. Marine Biotechnology 3:322-335.
Buchanan, J.T., C.G. Paniagua-Chavez, T.R. Tiersch, and R.K. Cooper. 2000. Considerations for Research-scale Manipulation of Oysters. National Shellfisheries Association Annual Meeting, Seattle, Washington, March 19-23. Journal of Shellfish Research 19:661. (Best Poster Award).
Buchanan, J.T., J.F. La Peyre, T.R. Tiersch, and R.K. Cooper. 2001. Optimization of Gene Delivery for Improved Oyster Health. International Chapter of the World Aquaculture Society Annual Meeting. Orlando, Florida, January 21-25. Book of Abstracts p. 94.
Buchanan, J T, T.C. Cheng, J.F. La Peyre, R.K. Cooper, and T.R. Tiersch. 2001. in vivo transfection of adult eastern oyster. Journal of the World Aquaculture Society 32:286-299.
![[Maryland Sea Grant]](../mdsg_logo.gif){kind=logo}
![[NOAA]](../noaa_logo.gif){kind=logo}
![[Virginia Sea Grant]](../vasg_logo.gif){kind=logo}