Download Maintenance of Transgenic BY

C-TBY2-014
Maintenance of Transgenic BY-2 Cell Suspension Cultures
Introduction
Tobacco GF11, GT16, GV7, and TBY2-AtRER1B are transgenic BY-2 cell lines
expressing Green Fluorescent Protein (GFP) fused with four different plant
proteins, AtFim1 (Sano et al. 2005), tubulin α (Kumagai et al. 2001), AtVAM3
(Kutsuna et al. 2003), and AtRer1B (Takeuchi et al. 2002), respectively. Because
the cellular target site of GFP fused proteins can be visualized by using a
fluorescence microscope, the transgenic BY2 cell lines are used for studying
detailed structures of plant cells. Our transgenic BY-2 cell lines have been
maintained in a modified Linsmaier and Skoog (mLS) medium in dark at 27°C
and subcultured at one-week intervals.
Materials and Methods
I.
Glassware and mLS medium are prepared as described in “Maintenance of
Tobacco BY-2 Cell Suspension Culture.”
II.
The transgenic BY-2 cell lines are maintained by using almost the same
method as the parent BY-2 cell line. An adequate amount of cell suspension
indicated in Table 1 is transferred 95 ml of fresh mLS medium in a 300-ml
Erlenmeyer flask for every subculturing.
References
Kumagai F, Yoneda A, Tomida T, Sano T, Nagata T, Hasezawa S (2001)
Fate of nascent microtubules organized at the M/G1 interface, as
visualized by synchronized tobacco BY-2 cells stably expressing
GFP-Tubulin: time-sequence observations of the reorganization of
cortical microtubules in living plant cells. Plant and Cell Physiology 42:
723-732
Kutsuna N, Kumagai F, Sato MH, Hasezawa S (2003) Three-dimensional
reconstruction of tubular structure of vacuolar membrane throughout
mitosis in living tobacco cells. Plant and Cell Physiology 44: 1045-1054
Sano T, Higaki T, Oda Y, Hayashi T, Hasezawa S (2005) Appearance of
actin microfilament ‘twin peaks’ in mitosis and their function in cell plate
formation, as visualized in tobacco BY-2 cells expressing GFP-Fimbrin.
Plant Journal 44: 595-605
Takeuchi M, Ueda T, Yahara N, Nakano A (2002) Arf1 GTPase plays roles in
the protein traffic between the endoplasmic reticulum and the Golgi
apparatus in tobacco and Arabidopsis cultured cells. Plant Journal 31:
499-515
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Table 1 Subculture of transgenic BY-2 cell suspension cultures
Cell line
Subculture volume (ml) *
BY-2 (parent cell line)
1.4–1.8
GF11
2.8–3.4
GT16
3.0–3.8
GV7
–
TBY2-AtRER1B
–
* Cell suspension is transferred to 95 ml of fresh mLS medium.
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