Genetic transformation of tobacco NT1 cells with Agrobacterium tumefaciens.
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.
Mayo
Kristin J
KJ
Biodesign Institute at Arizona State University, Center for Infectious Diseases and Vaccinology, Tempe, Arizona 85287-5401, USA.
Gonzales
Barbara J
BJ
Mason
Hugh S
HS
D016960:Agrobacterium tumefaciens; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006514:Hepatitis B Surface Antigens / Q000378:metabolism; D007612:Kanamycin / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics*
D006514:Hepatitis B Surface Antigens; D007612:Kanamycin