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Agrobacterium Protocols Volume 2 pp 435–451Cite as

Mammalian Cells

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 344))

Abstract

Agrobacterium most likely can transform virtually all known plant species, and experimental protocols for Agrobacterium-mediated genetic transformation of yet more plant species, ecotypes, and cultivars are published almost on a daily basis. Interestingly, the Agrobacterium host range is not limited to the plant kingdom, and it has been shown to transform many species of fungi and even prokaryotes. The ability of Agrobacterium to genetically transform HeLa cells further widens the range of potential hosts of Agrobacterium to include humans and perhaps other animal species. Furthermore, because mammalian cells significantly differ from plant cells, they provide a useful experimental system for identification and functional characterization of plant-specific factors involved in the transformation process. Here, we present basic procedures for transfection and Agrobacterium-mediated genetic transformation of mammalian cells. We also demonstrate the use of mammalian cells for studies of the cellular components of the genetic transformation pathway.

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References

  1. Gaudin, V., Vrain, T., and Jouanin, L. (1994). Bacterial genes modifying hormonal balances in plants. Plant Physiol. Biochem. 32, 11–29.

    CAS  Google Scholar 

  2. Bracha-Drori, K., Shichrur, K., Katz, A., et al. (2004). Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. Plant J. 40, 419–427.

    Article  PubMed  CAS  Google Scholar 

  3. Walter, M., Chaban, C., Schütze, K., et al. (2004). Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40, 428–438.

    Article  PubMed  CAS  Google Scholar 

  4. Bevan, M.W. (1984). Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12, 1811–1821.

    Article  Google Scholar 

  5. Draper, J., Scott, R., Armitage, P., and Walden, R. (eds.) (1988) Plant Genetic Transformation and Gene Expression, A Laboratory Manual. Blackwell Scientific Publications Ltd., London, UK.

    Google Scholar 

  6. Clough, S.J. and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.

    Article  PubMed  CAS  Google Scholar 

  7. Gelvin, S.B. (1998). The introduction and expression of transgenes in plants. Curr. Opin. Biotechnol. 9, 227–232.

    Article  PubMed  CAS  Google Scholar 

  8. Tzfira, T. and Citovsky, V. (2000). From host recognition to T-DNA integration: the function of bacterial and plant genes in the Agrobacterium-plant cell interaction. Mol. Plant Pathol. 1, 201–212.

    Article  PubMed  CAS  Google Scholar 

  9. Tzfira, T., Li, J., Lacroix, B., and Citovsky, V. (2004). Agrobacterium T-DNA integration: molecules and models. Trends Genet. 20, 375–383.

    Article  PubMed  CAS  Google Scholar 

  10. Tzfira, T. and Citovsky, V. (2002). Partners-in-infection: host proteins involved in the transformation of plant cells by Agrobacterium. Trends Cell Biol. 12, 121–129.

    Article  PubMed  CAS  Google Scholar 

  11. Gelvin, S.B. (2003). Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol. Mol. Biol. Rev. 67, 16–37.

    Article  PubMed  CAS  Google Scholar 

  12. Gelvin, S.B. (2000). Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 223–256.

    Article  PubMed  CAS  Google Scholar 

  13. Vergunst, A.C., Schrammeijer, B., den Dulk-Ras, A., de Vlaam, C.M.T., Regensburg-Tuink, T.J., and Hooykaas, P.J.J. (2000). VirB/D4-dependent protein translocation from Agrobacterium into plant cells. Science 290, 979–982.

    Article  PubMed  CAS  Google Scholar 

  14. Christie, P.J. and Vogel, J.P. (2000). Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol. 8, 354–360.

    Article  PubMed  CAS  Google Scholar 

  15. Ding, Z., Atmakuri, K., and Christie, P.J. (2003). The outs and ins of bacterial type IV secretion substrates. Trends Microbiol. 11, 527–535.

    Article  PubMed  CAS  Google Scholar 

  16. Cascales, E. and Christie, P.J. (2004). Definition of a bacterial type IV secretion pathway for a DNA substrate. Science 304, 1170–1173.

    Article  PubMed  CAS  Google Scholar 

  17. Kelly, B.A. and Kado, C.I. (2002). Agrobacterium-mediated T-DNA transfer and integration into the chromosome of Streptomyces lividans. Mol. Plant Pathol. 3, 125–134.

    Article  PubMed  CAS  Google Scholar 

  18. Piers, K.L., Heath, J.D., Liang, X., Stephens, K.M., and Nester, E.W. (1996). Agrobacterium tumefaciens-mediated transformation of yeast. Proc. Natl. Acad. Sci. USA 93, 1613–1618.

    Article  PubMed  CAS  Google Scholar 

  19. Bundock, P., den Dulk-Ras, A., Beijersbergen, A., and Hooykaas, P.J.J. (1995). Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J. 14, 3206–3214.

    PubMed  CAS  Google Scholar 

  20. Bundock, P. and Hooykaas, P.J.J. (1996). Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc. Natl. Acad. Sci. USA 93, 15,272–15,275.

    Article  PubMed  CAS  Google Scholar 

  21. de Groot, M.J., Bundock, P., Hooykaas, P.J.J., and Beijersbergen, A.G. (1998). Agrobacterium tumefaciens-mediated transformation of filamentous fungi [published erratum appears in Nat. Biotechnol. 16, 1074 (1998)]. Nat. Biotechnol. 16, 839–842.

    Article  PubMed  Google Scholar 

  22. Michielse, C.B., Salim, K., Ragas, P., et al. (2004). Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer. Mol. Genet. Genomics 271, 499–510.

    Article  PubMed  CAS  Google Scholar 

  23. Kunik, T., Tzfira, T., Kapulnik, Y., Gafni, Y., Dingwall, C., and Citovsky, V. (2001). Genetic transformation of HeLa cells by Agrobacterium. Proc. Natl. Acad. Sci. USA 98, 1871–1876.

    Article  PubMed  CAS  Google Scholar 

  24. Pelczar, P., Kalck, V., Gomez, D., and Hohn, B. (2004). Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells. EMBO Rep. 5, 632–637.

    Article  PubMed  CAS  Google Scholar 

  25. Tzfira, T., Frankmen, L., Vaidya, M., and Citovsky, V. (2003). Site-specific integration of Agrobacterium T-DNA via double-stranded intermediates. Plant Physiol. 133, 1011–1023.

    Article  PubMed  CAS  Google Scholar 

  26. Chilton, M.-D.M. and Que, Q. (2003). Targeted integration of T-DNA into the tobacco genome at double-strand breaks: new insights on the mechanism of T-DNA integration. Plant Physiol. 133, 956–965.

    Article  PubMed  CAS  Google Scholar 

  27. van Attikum, H. and Hooykaas, P.J.J. (2003). Genetic requirements for the targeted integration of Agrobacterium T-DNA in Saccharomyces cerevisiae. Nucleic Acids Res. 31, 826–832.

    Article  PubMed  Google Scholar 

  28. Tzfira, T., Vaidya, M., and Citovsky, V. (2001). VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity. EMBO J. 20, 3596–3607.

    Article  PubMed  CAS  Google Scholar 

  29. Tzfira, T., Vaidya, M., and Citovsky, V. (2002). Increasing plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis VIP1 gene. Proc. Natl. Acad. Sci. USA 99, 10,435–10,440.

    Article  PubMed  CAS  Google Scholar 

  30. Citovsky, V., Kapelnikov, A., Oliel, S., et al. (2004). Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro. J. Biol. Chem. 279, 29,528–29,533.

    Article  PubMed  CAS  Google Scholar 

  31. Britt, A.B. and May, G.D. (2003). Re-engineering plant gene targeting. Trends Plant Sci. 8, 90–95.

    Article  PubMed  CAS  Google Scholar 

  32. Pelczar, P., Kalck, V., and Kovalchuk, I. (2003). Different genome maintenance strategies in human and tobacco cells. J. Mol. Biol. 331, 771–779.

    Article  PubMed  CAS  Google Scholar 

  33. Felgner, P.L., Gadek, T.R., Holm, M., et al. (1987). Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA 84, 7413–7417.

    Article  PubMed  CAS  Google Scholar 

  34. Brewer, C.B. and Roth, M.G. (1991). A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin. J. Cell Biol. 114, 413–421.

    Article  PubMed  CAS  Google Scholar 

  35. Tzfira, T., Jensen, C.S., Wangxia, W., Zuker, A., Altman, A., and Vainstein, A. (1997). Transgenic Populus: a step-by-step protocol for its Agrobacterium-mediated transformation. Plant Mol. Biol. Rep. 15, 219–235.

    Article  CAS  Google Scholar 

  36. Ausubel, F.M., Brent, R., Kingston, R.E., et al. (1987). Current Protocols in Molecular Biology, Greene Publishing-Wiley Interscience, New York.

    Google Scholar 

  37. Hajdukiewicz, P., Svab, Z., and Maliga, P. (1994). The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25, 989–994.

    Article  PubMed  CAS  Google Scholar 

  38. Binns, A.N. and Thomashow, M.F. (1988). Cell biology of Agrobacterium infection and transformation of plants. Annu. Rev. Microbiol. 42, 575–606.

    Article  CAS  Google Scholar 

  39. Zambryski, P.C. (1989). Agrobacterium-plant cell DNA transfer. In: Mobile DNA (Berg, D.E. and Howe, M.M. eds.), American Society for Microbiology, Washington, DC, pp. 309–333.

    Google Scholar 

  40. Tinland, B. (1996). The integration of T-DNA into plant genomes. Trends Plant Sci. 1, 178–184.

    Article  Google Scholar 

  41. Gielen, J., Terryn, N., Villarroel, R., and Van Montagu, M. (1999). Complete nucleotide sequence of the T-DNA region of the plant tumour-inducing Agrobacterium tumefaciens Ti plasmid pTiC58. J. Exp. Bot. 50, 1421–1422.

    Article  CAS  Google Scholar 

  42. Ballas, N. and Citovsky, V. (1997). Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein. Proc. Natl. Acad. Sci. USA 94, 10,723–10,728.

    Article  PubMed  CAS  Google Scholar 

  43. Tzfira, T. and Citovsky, V. (2001). Comparison between nuclear import of nopaline-and octopine-specific VirE2 protein of Agrobacterium in plant and animal cells. Mol. Plant Pathol. 2, 171–176.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, The University of Michigan, Ann Arbor, MI

    Tzvi Tzfira

  2. Fertiseeds Ltd., 13A Building, K. Weizmann Science Park, Ness Ziona, 74140, Israel

    Talya Kunik

  3. Institute of Field and Garden Crops, Agricultural Research Organization, P.O. Box 6, Bet Dagan, 50250, Israel

    Yedidya Gafni

  4. Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY

    Vitaly Citovsky

Authors
  1. Tzvi Tzfira
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  2. Talya Kunik
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  3. Yedidya Gafni
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  4. Vitaly Citovsky
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Editor information

Editors and Affiliations

  1. Center for Plant Transformation, Plant Science Institute, Iowa State Universtiy, Ames, Iowa

    Kan Wang

  2. Department of Agronomy, Iowa State Universtiy, Ames, Iowa

    Kan Wang

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© 2006 Humana Press Inc., Totowa, NJ

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Tzfira, T., Kunik, T., Gafni, Y., Citovsky, V. (2006). Mammalian Cells. In: Wang, K. (eds) Agrobacterium Protocols Volume 2. Methods in Molecular Biology, vol 344. Humana Press. https://doi.org/10.1385/1-59745-131-2:435

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  • DOI: https://doi.org/10.1385/1-59745-131-2:435

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-843-0

  • Online ISBN: 978-1-59745-131-4

  • eBook Packages: Springer Protocols

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