Electroporated intact BY-2 tobacco culture cells as a model of transient expression study.

• Authors: Edyta Kościańska , Krzysztof Wypijewski
• Languages of publication: EN

Abstracts

EN

Transfer of foreign genes into plant cells can be accomplished by several methods: agrobacterium-mediated, microinjection, biolistic particle bombardment and electroporation. The last one is frequently used for transfection of plant protoplasts for transient gene expression. Electroporation is a simple procedure and allows transfecting a large number of cells at one time. Square wave-modulated porators are the most efficient for introducing expression cassettes into plant protoplasts. Based on a protocol developed by Wu & Feng (Plant Cell Reports, 1999, 18, 381-386), we optimized conditions for transfection of intact Nicotiana tabacum BY-2 cells using square wave-modulated electroporator. To simplify screening for transfected gene expression we used constructs with a GFP marker gene.Electroporation of cells in the presence of DNA has been widely used in recent years in molecular biology for studying transient gene expression. It consists in subjecting cells to an electric field, which forms pores in the lipid bilayer of the cell membrane, allowing DNA molecules to enter into the cytoplasm [1]. Pore formation is reversible and cell survival is maintained, thus such a method of introducing foreign DNA into cells is fast, simple, efficient, non-toxic and applicable to a great variety of cells. However, in spite of all its advantages electroporation has not been applied equally successfully in experiments with plant cells (except those with protoplasts) because of the cell wall. There are some earlier reports indicating that the cell wall does not prevent DNA molecules from being internalized [1-3]. In 1999 Wu and Feng [4] described an effective method of electroporation applicable to the intact plant cells. In this method plant cells are subjected to plasmolysis prior to electroporation. The modifications of these procedure are presented in this paper.

Keywords

EN

transfection; square-wave electroporator; BY-2 cells

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2001
• Volume: 48
• Issue: 3
• Pages: 657-661

Dates

published

2001

received

2001-01-22

revised

2001-04-2

accepted

2001-08-16

Contributors

author

Edyta Kościańska

• Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznań, Poland

author

Krzysztof Wypijewski

• Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznań, Poland

References

• 1. Lurquin, P.F. (1997) Mol. Biotechnol. 7, 5-35.
• 2. Wu, F.S. & Cahoon, A.B. (1995) Plant Sci. 104, 201-214.
• 3. Potrykus, I. (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 205-225.
• 4. Wu, F.S. & Feng, T.Y. (1999) Plant Cell Reports 18, 381-386.
• 5. Nagata, T., Nemoto, Y. & Hasezawa, S. (1992) Int. Rev. Cytol. 132, 1-30.
• 6. Murashige, T. & Skoog, F. (1962) Physiol. Plant. 15, 473-497.
• 7. Haseloff, J. & Amos, B. (1995) Trends Genet 11, 328-329.
• 8. Sambrook, J. & Russell, D.W. (2001). Molecular Cloning, 3rd edn., Cold Spring Harbor Laboratory Press.