Summary
The inability to stably introduce and express foreign genes has hampered basic research in molluscan species. We cultured cells from dissociated embryos of the Pacific oyster, Crassostrea gigas, and infected these primary cultures with pantropic retroviral vectors containing the envelope glycoprotein of vesicular stomatitis virus. Luciferase transgene expression mediated by different heterologous promoters was demonstrated for at least 9 d after infection of the cells. Surprisingly, the promoter reproducibly mediating the highest level of luciferase expression was the retroviral promoter (U3 region of long terminal repeat) from the Moloney murine leukemia virus. The infection efficiency using a low multiplicity of infection (0.05) was estimated by quantitative polymerase chain reaction to be between 0.1–0.5%. This system will facilitate studies of gene expression and regulation and should be widely applicable to other molluscan species.
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Boulo, V.; Cadoret, J. P.; Le Marrec, F.; Dorange, G.; Miahle, E. Transient expression of luciferase reporter gene after lipofection in oyster (Crassostrea gigas) primary cell cultures. Mol. Mar. Biol. Biotechnol. 5: 167–174; 1996.
Burns, J. C.; Friedmann, T.; Driever, D.; Burrascano, M.; Yee, J.-K. VSV-G pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and non-mammalian cells. Proc. Natl. Acad. Sci. USA 90:8033–8037; 1993.
Cadoret, J. P.; Boulo, V.; Gendreau, S.; Miahle, E. Promoters from Drosophila heat shock protein and cytomegalovirus drive transient expression of luciferase introduced by particle bombardment into embryos of the oyster Crassostrea gigas. J. Biotechnol. 56:183–189; 1997a.
Cadoret, J. P.; Gendreau, S.; Delecheneau, J. M.; Rousseau, C.; Miahle, E. Microinjection in bivalve eggs: application in genetics. Mol. Mar. Biol. Biotechnol. 6:72–77; 1997b.
Cadoret, J. P.; Debon, R; Cornudella, L; Lardans, V; Morvan, A.; Roch, P.; Boulo, V. Transient expression assays with the proximal promoter of a newly characterized actin gene from the oyster Crassostrea gigas. FEBS Lett., in press.
Goldberg, R. B.; Crain, W. R.; Ruderman, J. V., et al. DNA sequence organization in the genomes of five marine invertebrates. Chromosoma 51: 225–251; 1975.
Heid, C. A.; Steven, J.; Livak, K. J.; Williams, P. M. Real time quantitative PCR. Genome Res. 6:986–994; 1996.
Jordan, T. V.; Shike, H.; Boulo, V., et al. Pantropic retroviral vectors mediate somatic cell transformation and expression of foreign genes in dipteran insects. Insect Mol. Biol. 7:215–222; 1998.
Lu, J-K.; Burns, J. C.; Chen, T. T. Pantropic retroviral vector integration, expression, and germline transmission in medaka (Oryzias latipes). Mol. Mar. Biol. Biotechnol. 6:289–295; 1997.
Lu, J. K.; Chen, T. T.; Allen, S. K.; Matsubara, T.; Burns, J. C. Production of transgenic dwarf surfclams, Mulinia lateralis, with pantropic retroviral vectors. Proc. Natl. Acad. Sci. USA 93:3482–3486; 1996.
Matsubara, T.; Beeman, R. W.; Shike, H., et al. Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae. Proc. Natl. Acad. Sci. USA 93:6181–6185; 1996.
Powers, D. A.; Kirby, V. L.; Cole, T.; Hereford, L. Electroporation as an effective means of introducing DNA into abalone (Haliotis rufescens) embryos. Mol. Mar. Biol. Biotechnol. 4:369–376; 1995.
Que, X.; Kim, D.; Alagon, A., et al. Pantropic retroviral vectors mediate stable gene transfer and expression in Entamoeba histolytica. Mol. Biochem. Parasitol. 99:237–245; 1999.
Teysset, L.; Burns, J. C.; Shike, H.; Sullivan, B. L.; Bucheton, A.; Terzian, C. A Moloney murine leukemia virus-based retroviral vector pseudotyped by the insect retroviral gypsy envelope can infect Drosophila cells. J. Virol. 72:853–856; 1998.
Wang, S.; Beattie, G.; Hayek, A.; Levine, F. Transformation of human primary fibroblasts by pseudotyped retroviral vectors expressing multiple dominant oncogenes from an inducible promoter. Gene 182:145–150; 1996.
Xu, L.; Wolf, J. A.; Friedmann, T. Factors affecting long-term stability of Moloney murine leukemia virus-based vectors. Virology 171:331–341; 1989.
Yee, J.-K.; Friedmann, T.; Burns, J. C. Generation of high-titer pseudotyped retroviral vectors with very broad host range. Methods Cell Biol. 43: 99–112; 1994.
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Boulo, V., Cadoret, JP., Shike, H. et al. Infection of cultured embryo cells of the pacific oyster, Crassostrea gigas, by pantropic retroviral vectors. In Vitro Cell.Dev.Biol.-Animal 36, 395–399 (2000). https://doi.org/10.1290/1071-2690(2000)036<0395:IOCECO>2.0.CO;2
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DOI: https://doi.org/10.1290/1071-2690(2000)036<0395:IOCECO>2.0.CO;2