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Simultaneous monitoring of independent gene expression patterns in two types of cocultured fibroblasts with different color-emitting luciferases.

Noguchi T, Ikeda M, Ohmiya Y, Nakajima Y - BMC Biotechnol. (2008)

Bottom Line: The circadian rhythms of these cell lines are independent, oscillating following their intrinsic circadian phases, even when cocultured.Furthermore, the independent rhythms were synchronized by medium change as an external stimulus.Using this system, we successfully monitored independent gene expression patterns in two lines of cocultured fibroblasts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell Dynamics Research Group, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan. t-noguchi@aist.go.jp

ABSTRACT

Background: Luciferase assay systems enable the real-time monitoring of gene expression in living cells. We have developed a dual-color luciferase assay system in which the expression of multiple genes can be tracked simultaneously using green- and red-emitting beetle luciferases. We have applied the system to monitoring independent gene expressions in two types of cocultured fibroblasts in real time.

Results: Two Rat-1 cell lines were established that stably express either green- or red-emitting luciferases under the control of the mBmal1 promoter, a canonical clock gene. We cocultured these cell lines, and gene expression profiles in both were monitored simultaneously. The circadian rhythms of these cell lines are independent, oscillating following their intrinsic circadian phases, even when cocultured. Furthermore, the independent rhythms were synchronized by medium change as an external stimulus.

Conclusion: Using this system, we successfully monitored independent gene expression patterns in two lines of cocultured fibroblasts.

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Related in: MedlinePlus

Analysis of the circadian phases of Bmal1–GR and Bmal1–RED cells cocultured with direct contact. The bioluminescence activities of Bmal1–GR (A), Bmal1–RED (B), and cocultures of these cells (C) were measured. The luciferase activities are shown on the Y axis and the time after starting measurement of the bioluminescence is shown on the X axis. These cultures showed clear circadian rhythms. D: Average peak times after the start of DEX treatment of Bmal1–GR cells in monoculture (filled diamonds), in coculture (open diamonds), Bmal1–RED cells in monoculture (filled circles) and in coculture (open circles). The number of samples is shown under the symbols. The bars show the SEM of the data. The phases of each cell line gradually became separated in both cocultures and monocultures. E: Amplitude-damping rates of Bmal1–GR and Bmal1–RED cells in monoculture and coculture are shown. There were no significant differences in damping rates between cell lines or between monocultures and cocultures (P > 0.05, Mann-Whitney U test).
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Figure 3: Analysis of the circadian phases of Bmal1–GR and Bmal1–RED cells cocultured with direct contact. The bioluminescence activities of Bmal1–GR (A), Bmal1–RED (B), and cocultures of these cells (C) were measured. The luciferase activities are shown on the Y axis and the time after starting measurement of the bioluminescence is shown on the X axis. These cultures showed clear circadian rhythms. D: Average peak times after the start of DEX treatment of Bmal1–GR cells in monoculture (filled diamonds), in coculture (open diamonds), Bmal1–RED cells in monoculture (filled circles) and in coculture (open circles). The number of samples is shown under the symbols. The bars show the SEM of the data. The phases of each cell line gradually became separated in both cocultures and monocultures. E: Amplitude-damping rates of Bmal1–GR and Bmal1–RED cells in monoculture and coculture are shown. There were no significant differences in damping rates between cell lines or between monocultures and cocultures (P > 0.05, Mann-Whitney U test).

Mentions: Equal numbers of Bmal1–GR and Bmal1–RED cells were mixed and grown in a dish. As controls, monocultures of either Bmal1–GR or Bmal1–RED cells were cultured individually (Fig. 3A, B). The cells were stimulated by replacing the culture medium with medium containing DEX. Both cell lines showed clear circadian rhythms in their luciferase activities, even when cocultured with direct contact (Fig. 3C). The peak times after the start of DEX treatment and the rate of damping in amplitude were analyzed statistically (Fig. 3D, E). The peak time of Bmal1–GR cells advanced to that of Bmal1–RED cells at peak 1 but it was reversed at peak 2 and the difference was enlarged at peak 3. There were no significant differences in peak times or amplitude-damping rates between the monoculture and coculture systems (P > 0.05, Mann-Whitney U test). Thus, the independence of the circadian rhythms of Rat-1 cells was confirmed using this dual-color luciferase assay system.


Simultaneous monitoring of independent gene expression patterns in two types of cocultured fibroblasts with different color-emitting luciferases.

Noguchi T, Ikeda M, Ohmiya Y, Nakajima Y - BMC Biotechnol. (2008)

Analysis of the circadian phases of Bmal1–GR and Bmal1–RED cells cocultured with direct contact. The bioluminescence activities of Bmal1–GR (A), Bmal1–RED (B), and cocultures of these cells (C) were measured. The luciferase activities are shown on the Y axis and the time after starting measurement of the bioluminescence is shown on the X axis. These cultures showed clear circadian rhythms. D: Average peak times after the start of DEX treatment of Bmal1–GR cells in monoculture (filled diamonds), in coculture (open diamonds), Bmal1–RED cells in monoculture (filled circles) and in coculture (open circles). The number of samples is shown under the symbols. The bars show the SEM of the data. The phases of each cell line gradually became separated in both cocultures and monocultures. E: Amplitude-damping rates of Bmal1–GR and Bmal1–RED cells in monoculture and coculture are shown. There were no significant differences in damping rates between cell lines or between monocultures and cocultures (P > 0.05, Mann-Whitney U test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2375886&req=5

Figure 3: Analysis of the circadian phases of Bmal1–GR and Bmal1–RED cells cocultured with direct contact. The bioluminescence activities of Bmal1–GR (A), Bmal1–RED (B), and cocultures of these cells (C) were measured. The luciferase activities are shown on the Y axis and the time after starting measurement of the bioluminescence is shown on the X axis. These cultures showed clear circadian rhythms. D: Average peak times after the start of DEX treatment of Bmal1–GR cells in monoculture (filled diamonds), in coculture (open diamonds), Bmal1–RED cells in monoculture (filled circles) and in coculture (open circles). The number of samples is shown under the symbols. The bars show the SEM of the data. The phases of each cell line gradually became separated in both cocultures and monocultures. E: Amplitude-damping rates of Bmal1–GR and Bmal1–RED cells in monoculture and coculture are shown. There were no significant differences in damping rates between cell lines or between monocultures and cocultures (P > 0.05, Mann-Whitney U test).
Mentions: Equal numbers of Bmal1–GR and Bmal1–RED cells were mixed and grown in a dish. As controls, monocultures of either Bmal1–GR or Bmal1–RED cells were cultured individually (Fig. 3A, B). The cells were stimulated by replacing the culture medium with medium containing DEX. Both cell lines showed clear circadian rhythms in their luciferase activities, even when cocultured with direct contact (Fig. 3C). The peak times after the start of DEX treatment and the rate of damping in amplitude were analyzed statistically (Fig. 3D, E). The peak time of Bmal1–GR cells advanced to that of Bmal1–RED cells at peak 1 but it was reversed at peak 2 and the difference was enlarged at peak 3. There were no significant differences in peak times or amplitude-damping rates between the monoculture and coculture systems (P > 0.05, Mann-Whitney U test). Thus, the independence of the circadian rhythms of Rat-1 cells was confirmed using this dual-color luciferase assay system.

Bottom Line: The circadian rhythms of these cell lines are independent, oscillating following their intrinsic circadian phases, even when cocultured.Furthermore, the independent rhythms were synchronized by medium change as an external stimulus.Using this system, we successfully monitored independent gene expression patterns in two lines of cocultured fibroblasts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cell Dynamics Research Group, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan. t-noguchi@aist.go.jp

ABSTRACT

Background: Luciferase assay systems enable the real-time monitoring of gene expression in living cells. We have developed a dual-color luciferase assay system in which the expression of multiple genes can be tracked simultaneously using green- and red-emitting beetle luciferases. We have applied the system to monitoring independent gene expressions in two types of cocultured fibroblasts in real time.

Results: Two Rat-1 cell lines were established that stably express either green- or red-emitting luciferases under the control of the mBmal1 promoter, a canonical clock gene. We cocultured these cell lines, and gene expression profiles in both were monitored simultaneously. The circadian rhythms of these cell lines are independent, oscillating following their intrinsic circadian phases, even when cocultured. Furthermore, the independent rhythms were synchronized by medium change as an external stimulus.

Conclusion: Using this system, we successfully monitored independent gene expression patterns in two lines of cocultured fibroblasts.

Show MeSH
Related in: MedlinePlus