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All-optical regulation of gene expression in targeted cells.

Wang Y, He H, Li S, Liu D, Lan B, Hu M, Cao Y, Wang C - Sci Rep (2014)

Bottom Line: Recently, various approaches based on extra-engineered light-sensitive proteins have been developed to provide optogenetic actuators for gene expression.Complicated biomedical techniques including exogenous genes engineering, transfection, and material delivery are needed.Intrinsic or exogenous genes can be activated by a Ca(2+)-sensitive transcription factor nuclear factor of activated T cells (NFAT) driven by a short flash of femtosecond-laser irradiation.

View Article: PubMed Central - PubMed

Affiliation: Ultrafast Laser Laboratory, Key Laboratory of Optoelectronic Information Technology (Ministry of Education), College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, 300072, P.R. China.

ABSTRACT
Controllable gene expression is always a challenge and of great significance to biomedical research and clinical applications. Recently, various approaches based on extra-engineered light-sensitive proteins have been developed to provide optogenetic actuators for gene expression. Complicated biomedical techniques including exogenous genes engineering, transfection, and material delivery are needed. Here we present an all-optical method to regulate gene expression in targeted cells. Intrinsic or exogenous genes can be activated by a Ca(2+)-sensitive transcription factor nuclear factor of activated T cells (NFAT) driven by a short flash of femtosecond-laser irradiation. When applied to mesenchymal stem cells, expression of a differentiation regulator Osterix can be activated by this method to potentially induce differentiation of them. A laser-induced "Ca(2+)-comb" (LiCCo) by multi-time laser exposure is further developed to enhance gene expression efficiency. This noninvasive method hence provides an encouraging advance of gene expression regulation, with promising potential of applying in cell biology and stem-cell science.

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Laser-regulated gene expression in HeLa cells.(a). TNF-α can be detected with or without ionomycin and Ca2+ treatment in HeLa cells as an endogenous gene tested by Western Blot. Tubulin: internal control in Western Blot. (b). Immunofluorescence microscopy of TNF-α in HeLa cells. Right: Upregulation of TNF-α in the cells with laser treatment (n = 40 cells in each experiment). The expression is more efficient if the laser-exposure duration is longer. Error bar: standard error of the mean. (c). Demonstration of NFAT-linked luciferase reporter plasmid that was transfected into HeLa cells. (d). Immunofluorescence microscopy of luciferase in HeLa cells. Around 14% cells were transfected with NFAT-luciferase plasmid. If luciferase was expressed, there would be bright fluorescence in the cells indicated by yellow arrows. Right: The expression efficiency of luciferase by the optical method is close to the transfection efficiency determined by ionomycin and Ca2+-injection treatment. Error bar: standard error of the mean. Bar: 50 μm.
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f2: Laser-regulated gene expression in HeLa cells.(a). TNF-α can be detected with or without ionomycin and Ca2+ treatment in HeLa cells as an endogenous gene tested by Western Blot. Tubulin: internal control in Western Blot. (b). Immunofluorescence microscopy of TNF-α in HeLa cells. Right: Upregulation of TNF-α in the cells with laser treatment (n = 40 cells in each experiment). The expression is more efficient if the laser-exposure duration is longer. Error bar: standard error of the mean. (c). Demonstration of NFAT-linked luciferase reporter plasmid that was transfected into HeLa cells. (d). Immunofluorescence microscopy of luciferase in HeLa cells. Around 14% cells were transfected with NFAT-luciferase plasmid. If luciferase was expressed, there would be bright fluorescence in the cells indicated by yellow arrows. Right: The expression efficiency of luciferase by the optical method is close to the transfection efficiency determined by ionomycin and Ca2+-injection treatment. Error bar: standard error of the mean. Bar: 50 μm.

Mentions: Here we measured the expression of tumor necrosis factor alpha (TNF-α) in HeLa cells (Fig. 2a), an intrinsic and classic downstream gene of NFAT that plays an important role in immune response24, to verify our proposed scheme of laser-regulated gene expression. In experiments, HeLa cells were selected randomly and exposed for 0.1 s and 0.3 s respectively to the femtosecond laser. As shown in Fig. 2b, significant upregulation of TNF-α can be found in laser-treated cells (n = 20 cells in each experiment) after 6-hour incubation. The expression of TNF-α in cells exposed longer is more abundant because the NFAT activation can be more effective under intense laser stimulation.


All-optical regulation of gene expression in targeted cells.

Wang Y, He H, Li S, Liu D, Lan B, Hu M, Cao Y, Wang C - Sci Rep (2014)

Laser-regulated gene expression in HeLa cells.(a). TNF-α can be detected with or without ionomycin and Ca2+ treatment in HeLa cells as an endogenous gene tested by Western Blot. Tubulin: internal control in Western Blot. (b). Immunofluorescence microscopy of TNF-α in HeLa cells. Right: Upregulation of TNF-α in the cells with laser treatment (n = 40 cells in each experiment). The expression is more efficient if the laser-exposure duration is longer. Error bar: standard error of the mean. (c). Demonstration of NFAT-linked luciferase reporter plasmid that was transfected into HeLa cells. (d). Immunofluorescence microscopy of luciferase in HeLa cells. Around 14% cells were transfected with NFAT-luciferase plasmid. If luciferase was expressed, there would be bright fluorescence in the cells indicated by yellow arrows. Right: The expression efficiency of luciferase by the optical method is close to the transfection efficiency determined by ionomycin and Ca2+-injection treatment. Error bar: standard error of the mean. Bar: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061554&req=5

f2: Laser-regulated gene expression in HeLa cells.(a). TNF-α can be detected with or without ionomycin and Ca2+ treatment in HeLa cells as an endogenous gene tested by Western Blot. Tubulin: internal control in Western Blot. (b). Immunofluorescence microscopy of TNF-α in HeLa cells. Right: Upregulation of TNF-α in the cells with laser treatment (n = 40 cells in each experiment). The expression is more efficient if the laser-exposure duration is longer. Error bar: standard error of the mean. (c). Demonstration of NFAT-linked luciferase reporter plasmid that was transfected into HeLa cells. (d). Immunofluorescence microscopy of luciferase in HeLa cells. Around 14% cells were transfected with NFAT-luciferase plasmid. If luciferase was expressed, there would be bright fluorescence in the cells indicated by yellow arrows. Right: The expression efficiency of luciferase by the optical method is close to the transfection efficiency determined by ionomycin and Ca2+-injection treatment. Error bar: standard error of the mean. Bar: 50 μm.
Mentions: Here we measured the expression of tumor necrosis factor alpha (TNF-α) in HeLa cells (Fig. 2a), an intrinsic and classic downstream gene of NFAT that plays an important role in immune response24, to verify our proposed scheme of laser-regulated gene expression. In experiments, HeLa cells were selected randomly and exposed for 0.1 s and 0.3 s respectively to the femtosecond laser. As shown in Fig. 2b, significant upregulation of TNF-α can be found in laser-treated cells (n = 20 cells in each experiment) after 6-hour incubation. The expression of TNF-α in cells exposed longer is more abundant because the NFAT activation can be more effective under intense laser stimulation.

Bottom Line: Recently, various approaches based on extra-engineered light-sensitive proteins have been developed to provide optogenetic actuators for gene expression.Complicated biomedical techniques including exogenous genes engineering, transfection, and material delivery are needed.Intrinsic or exogenous genes can be activated by a Ca(2+)-sensitive transcription factor nuclear factor of activated T cells (NFAT) driven by a short flash of femtosecond-laser irradiation.

View Article: PubMed Central - PubMed

Affiliation: Ultrafast Laser Laboratory, Key Laboratory of Optoelectronic Information Technology (Ministry of Education), College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, 300072, P.R. China.

ABSTRACT
Controllable gene expression is always a challenge and of great significance to biomedical research and clinical applications. Recently, various approaches based on extra-engineered light-sensitive proteins have been developed to provide optogenetic actuators for gene expression. Complicated biomedical techniques including exogenous genes engineering, transfection, and material delivery are needed. Here we present an all-optical method to regulate gene expression in targeted cells. Intrinsic or exogenous genes can be activated by a Ca(2+)-sensitive transcription factor nuclear factor of activated T cells (NFAT) driven by a short flash of femtosecond-laser irradiation. When applied to mesenchymal stem cells, expression of a differentiation regulator Osterix can be activated by this method to potentially induce differentiation of them. A laser-induced "Ca(2+)-comb" (LiCCo) by multi-time laser exposure is further developed to enhance gene expression efficiency. This noninvasive method hence provides an encouraging advance of gene expression regulation, with promising potential of applying in cell biology and stem-cell science.

Show MeSH
Related in: MedlinePlus