Abstract
This protocol is used to produce stably transformed tobacco (Nicotiana tabacum) NT1 cell lines, using Agrobacterium tumefaciens–mediated DNA delivery of a binary vector containing a gene encoding hepatitis B surface antigen and a gene encoding the kanamycin selection marker. The NT1 cultures, at the appropriate stage of growth, are inoculated with A. tumefaciens containing the binary vector. A 3-day cocultivation period follows, after which the cultures are rinsed and placed on solid selective medium. Transformed colonies ('calli') appear in approximately 4 weeks; they are subcultured until adequate material is obtained for analysis of antigen production. 'Elite' lines are selected based on antigen expression and growth characteristics. The time required for the procedure from preparation of the plant cell materials to callus development is approximately 5 weeks. Growth of selected calli to sufficient quantities for antigen screening may require 4–6 weeks beyond the initial selection. Creation of the plasmid constructs, transformation of the A. tumefaciens line, and ELISA and Bradford assays to assess protein production require additional time.
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Acknowledgements
The authors thank L. Richter, M. Smith and Z. Huang for development and optimization of the ELISA protocol. This work was supported by grants R01 AI042836 from the National Institutes of Health and BES0109936 from National Science Foundation to H.S.M.
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B.J.G., preparation of screening procedure and sections related to screening and analysis. K.J.M., preparation of plant transformation procedure, preliminary text and photos. H.S.M., editing of text, editorial communications and advisor.
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Mayo, K., Gonzales, B. & Mason, H. Genetic transformation of tobacco NT1 cells with Agrobacterium tumefaciens. Nat Protoc 1, 1105–1111 (2006). https://doi.org/10.1038/nprot.2006.176
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DOI: https://doi.org/10.1038/nprot.2006.176
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