Limits...
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.

Show MeSH

Related in: MedlinePlus

Licco method can enhance the efficiency of gene expression.(a). Ca2+ spikes by Licco method. The frequency of Licco can be determined by the interval between two laser exposures and the duration of each Ca2+ spike. Red arrow: laser-exposure event. (b). Enhanced gene expression by Licco method with 6 spikes (n = 25 cells in each experiment). The femtosecond laser was working at 35 mW and each time the exposure duration is 0.15 s. Error bar: standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4061554&req=5

f4: Licco method can enhance the efficiency of gene expression.(a). Ca2+ spikes by Licco method. The frequency of Licco can be determined by the interval between two laser exposures and the duration of each Ca2+ spike. Red arrow: laser-exposure event. (b). Enhanced gene expression by Licco method with 6 spikes (n = 25 cells in each experiment). The femtosecond laser was working at 35 mW and each time the exposure duration is 0.15 s. Error bar: standard error of the mean.

Mentions: This method was then furthered to improve the gene expression efficiency which can be affected by Ca2+ oscillation in cells34. We designed laser induced “Ca2+-comb” (Licco) in cells by treating them with multi-time femtosecond-laser irradiation to stimulate Ca2+ spikes. Each time after optical stimulation, Ca2+ was released from Ca2+ store at first and then slowly taken up by cells again until next stimulation. In this way, the “Licco” can mimic cellular Ca2+ oscillation (Supplementary Fig. S4) in cells. There are two critical factors to design Licco. First, the interval between two laser exposures should be longer than the duration of Ca2+ spike. To induce high-level but short-duration Ca2+ spikes, the exposure duration was then tuned to be 0.15 s with 35-mW power. Second, the times of laser exposure should not exceed the safety condition, which was found in our experiments that most cells (>95% in 30 cells) would keep good membrane integrity after as more as 10-time laser exposure (each for 0.15 s). Here a 6-spike Licco was performed in MSCs as shown in Fig. 4a. The fluorescence intensity decreased along each spike because the cell did not rest enough after each exposure to generate resembling Ca2+ release and the fluorescence emission efficiency of fluorophores as well as their re-combination with Ca2+ was also decreasing. This Licco method was then used to enhance gene expression in MSCs as shown in Fig. 4b (n = 90 cells in each experiment). The gene expression efficiency was improved, probably because the activation of the gene transcription mediated by laser-induced Ca2+ was enhanced.


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)

Licco method can enhance the efficiency of gene expression.(a). Ca2+ spikes by Licco method. The frequency of Licco can be determined by the interval between two laser exposures and the duration of each Ca2+ spike. Red arrow: laser-exposure event. (b). Enhanced gene expression by Licco method with 6 spikes (n = 25 cells in each experiment). The femtosecond laser was working at 35 mW and each time the exposure duration is 0.15 s. Error bar: standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Licco method can enhance the efficiency of gene expression.(a). Ca2+ spikes by Licco method. The frequency of Licco can be determined by the interval between two laser exposures and the duration of each Ca2+ spike. Red arrow: laser-exposure event. (b). Enhanced gene expression by Licco method with 6 spikes (n = 25 cells in each experiment). The femtosecond laser was working at 35 mW and each time the exposure duration is 0.15 s. Error bar: standard error of the mean.
Mentions: This method was then furthered to improve the gene expression efficiency which can be affected by Ca2+ oscillation in cells34. We designed laser induced “Ca2+-comb” (Licco) in cells by treating them with multi-time femtosecond-laser irradiation to stimulate Ca2+ spikes. Each time after optical stimulation, Ca2+ was released from Ca2+ store at first and then slowly taken up by cells again until next stimulation. In this way, the “Licco” can mimic cellular Ca2+ oscillation (Supplementary Fig. S4) in cells. There are two critical factors to design Licco. First, the interval between two laser exposures should be longer than the duration of Ca2+ spike. To induce high-level but short-duration Ca2+ spikes, the exposure duration was then tuned to be 0.15 s with 35-mW power. Second, the times of laser exposure should not exceed the safety condition, which was found in our experiments that most cells (>95% in 30 cells) would keep good membrane integrity after as more as 10-time laser exposure (each for 0.15 s). Here a 6-spike Licco was performed in MSCs as shown in Fig. 4a. The fluorescence intensity decreased along each spike because the cell did not rest enough after each exposure to generate resembling Ca2+ release and the fluorescence emission efficiency of fluorophores as well as their re-combination with Ca2+ was also decreasing. This Licco method was then used to enhance gene expression in MSCs as shown in Fig. 4b (n = 90 cells in each experiment). The gene expression efficiency was improved, probably because the activation of the gene transcription mediated by laser-induced Ca2+ was enhanced.

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