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light-switchable transactivator Basic part

Usage and Biology:

Figure1.The_structure_of_GVAPO

GVAPO is a synthetic light-switch transgene system, the transactivator binds promoters upon blue-light exposure and rapidly initiates transcription of target transgenes in mammalian cells and in mice. this transgene system provides a robust and convenient way to spatiotemporally control gene expression and can be used to manipulate many biological processes in living systems with minimal perturbation. Vivid (VVD), the smallest light-oxygen-voltage (LOV) domain–containing protein, forms a rapidly exchanging dimer upon blue-light activation. This part utilizes the DNA-binding property of a Gal4(65)-VVD fusion protein would be light-switchable, as light should induce dimerization of the fusion protein, enhance binding to the UASG sequence and activate transcription and removing the light should result in gradual dissociation of the dimers, DNA dissociation and inactivation.

Figure2.Light-dependent activation of Fluc reporters based on GAVP with different mutations to enhance dimerization

Test their light-dependent impact on transcriptional activity of a firefly luciferase (Fluc) reporter driven by Gal4 binding sites upstream of a TATA box after transient transfection in HEK293 cells, illumination with 0.84 W m−2 460 nm peak light from an LED lamp for 22 h and measurement of expression. Transactivators containing the p65 activation domain (GAVP) or the VP16 activation domain (GAVV) both showed marked light-induced reporter gene transcription, but the GAVP transactivator resulted in much greater gene expression under light exposure conditions. Mutation of Cys108 in VVD to serine blocked light-inducible gene expression as expected13. Mutation of Cys71 to valine in VVD is known to enhance the stability of the light-induced VVD dimer14, and based on the crystal structure of VVD13 we hypothesized that mutating Gln56 of VVD to lysine would form a salt bridge with Asp68 of the other VVD protein and additionaly stabilize the dimer. Both dimer-enhancing mutations, C71V and N56K, in the VVD domain decreased reporter gene expression in the dark, whereas the N56K,C71V double mutant (optimized GAVP (GAVPO)) additionally decreased the background gene expression to a minimal level. So We used GAVPO in all subsequent studies, and we referred to the gene promoter system based on GAVPO as the light-on (LightOn) system.

Figure3.Comparison of induction levels of Fluc reporters driven by GAVV and the conventional CMV driven vectors.

DNA binding domain, the VVD light sensor and VP16 transactivation domain. Experimental conditions were as described for Figure.2. The data were normalized to the Fluc expression level of the pcDNA3.1-Fluc vector under dark condition. Error bars, mean ± s.e.m. (n = 4) from the same experiment.

Design Notes

How can we coupled this light-switch system with gene coded for the cytokines, which may stay in the cytosol or secrete to the outside of the cells. Whether changing another kind of cells can affect the effect of this system.

Source

This light-switch transgene system is form the lab of Xue Wang, Xianjun Chen & Yi Yang, they published Spatiotemporal control of gene expression by a light-switchable transgene system in Nature Methods 2012.

SequenceAndFeatures

References

Wang X , Chen X , Yang Y . Spatiotemporal control of gene expression by a light-switchable transgene system[J]. Nature Methods, 2012, 9(3):266-269.