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Abstracts
Workgroup: Genetics and Oyster Populations
Creation of an oyster cell line in C. virginica
Principal Investigator(s):
Jane C. Burns, University of California-San Diego, School of Medicine, jcburns@ucsd.edu
CO-INVESTIGATOR and AFFILIATION: Carolyn Friedman, University of California-Davis
CO-INVESTIGATOR and AFFILIATION: Carolyn Friedman, University of California-Davis
Funding Period: 1998
It was the goal of this project to assist the aquaculture industry through creation of a transformed C. virginica oyster cell line to facilitate studies focused on isolation and cultivation of oyster pathogens and oyster molecular biology. To achieve this goal, we proposed to develop modified retroviral vectors that contain proto-oncogenes as agents for cellular transformation. The specific goals of the project were:
- to design retroviral vectors that express a variety of proto-oncogenes from appropriate promoter elements that express in oyster cells,
- to infect primary cultures established from both oyster hearts and from enzymatically disrupted early embryos with the transforming retroviral vectors,
- to test resulting cell cultures for immortalization
- to test the cultivation of oyster pathogens in the immortalized oyster cell line
1) Retroviral vectors expressing the SV40 large T antigen and h-ras genes were constructed and used to infect cultured oyster heart cells. Despite PCR evidence that the virus was present in the cells, no expression could be detected by IF staining or Western blotting with appropriate antibodies.
2) Oyster heart infection with retroviral vectors: Infection of cultured heart cells with the pantropic vector expressing luciferase under the control of the LTR promoter yielded detectable luciferase activity that decreased as the cells began dying in culture. Although the dissociated heart cells could be maintained in culture for 1-2 months, the cells appear to stop dividing at the end of the first week in culture.
Hemoylmph analysis: To aid in the formulation of media to nutritionally support oyster cells in culture, we analysed the hemolymph components from Crassostrea virginica and C. gigas. We measured DNA synthesis in primary cultures incubated in different media and at different temperatures to optimize culture conditions.
Pooled hemolymph (2-3 oysters/pool) was obtained by cardiac puncture. Hemocytes were removed by centrifugation (1,000 x g) and the supernatants stored at -70ƒC. Analysis of hemolymph components included free amino acids, organic acids, carbohydrates, metals, electrolytes, pH and osmolality. Cultures of heart and embryos were established according to published methods (Boulo et al., 1996 and 2000). DNA synthesis in cultured cells was assessed by 3H-thymidine uptake.Two media formulations based on published data were compared: 1) KS medium (based on Kleinschuster and Swink 1993): L-15 adjusted to 750 mOsm with synthetic sea salts, and supplemented with amino acids, lipids, carbohydrates, vitamins, 10% fetal calf serum (FCS), and 10% C. virginica hemolymph, 2) 2X L-15 (Boulo et al., 1996) adjusted to 750 mOsm with NaCl plus 10% FCS.
Selected components that were lower in the media than in the pooled hemolymph samples are shown below:
Table 1. C. virginica hemolymph and media analysis expressed as mean values +/- S.D; NA= not available.
Component | C. virginica hemolymph | KS medium | 2X L-15/10%FBS | |
taurine | 195.9+/-12.1 mg/L | 51.9 | NA | |
proline | 90.5+/-3.7 | 34.2 | NA | |
calcium | 40.7+/-6.2 | 19.8 | 9.3 | |
strontium | 4.7+/-0.9 | 3.0 | 0.1 | |
boron | 4.4+/-0.7 | NA | 0.1 | |
zinc | 1.3+/-1.3 | NA | 0.1 | |
pH ranged from 6.4-6.9. | ||||
Different media formulations were made based on the above observations. We used retroviral vector infection of cultured heart cells with a luciferase vector as our assessment of cell division in the culture. It had been noted that luciferase activity following infection with the vector, LLRNL, directly correlated with 3H-thymidine results. Therefore, we examined the possibility of using luciferase activity as a surrogate marker for cell division following vector infection. In the Figure, we have two basal media comparisons (M-6 and M-1) with various additives to M-6. None of the differences were statistically significant, suggesting that none of the different formulations had a dramatic effect on cell division.
Summary: Despite the ability to infect and express foreign genes in cultured oyster cells, we were unsuccessful in creating a C. virginica immortalized cell line. The block to cell division in these cultured cells remains unclear. Attempts to manipulate the medium based on analysis of hemoylymph did not result in improved cell division in vitro.
Update: Based on these experiences, our group decided to focus on a naturally occurring neoplasia of mussel cells (Mytilus trossulus) as a more promising avenue to a molluscan cell line. This work is currently funded by California SeaGrant (R/A-119). Our first goal in this project is to determine if the hemolymph cells derive from malignant transformation of host mussel cells or whether they are "transplanted" from genetically non-identical methods. We are pursuing molecular markers for the neoplastic cells that will allow us to identify them after transplantation in the laboratory into healthy, recipient mussels.
PROJECT PUBLICATIONS AND PRESENTATIONS:
Boulo V, Cadoret JP, Shike H, Shimizu C, Miyanohara A, Burns JC. Infection of cultured embryo cells of the Pacific oyster, Crassostrea gigas, by pantropic retroviral vectors. In Vitro Cell. Devel. Biol. 36:395-399, 2000.
Burns JC, Shimizu C, Boulo V, Shike H. Pantropic retroviral vectors for gene transfer into invertebrate cells. Presented at the Society for In Vitro Biology, San Diego, CA June 2000; In Vitro Cell. Develop. Biol. 36:12-A, 2000.
Boulo V, Moore JD, Shimizu C, Friedmann CS, Burns JC. Infection of primary cultured cells from two oyster species by pantropic retroviral vectors. Presented at the Society for In Vitro Biology, San Diego, CA June 2000; In Vitro Cell. Develop. Biol. 36:37A, 2000.
Burns JC, Shimizu C, Shike H. Pantropic retroviral vectors for gene transfer in aquaculture species. Presented at the World Aquaculture Society Meeeting, Nice, France, May 2000.
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