The Combination

Our ultimate goal is to combine the spatial resolution of the light system with the specificity of our viral vector to generate patterns in a homogeneous cell culture. Since cells that are not illuminated with the inducing light should not express the mCAT-1 receptor, they should not get infected by viral particles.

Light Induced Receptor

For light induced gene transfer with our viral vector we cloned mCAT-1, the receptor our viral vector is specific for, into the response plasmid for the blue light system (plasmid p14ls_003 coding for mCAT-1-HA-P2A-mCherry under the control of the Gal4 upstream activating sequence and a minimal CMV promoter) or the red light system (p14rz_002 coding for mCAT-1-HA-P2A-mCherry under the control of the tet operon and a minimal CMV promoter). These P2A sequence between the mCAT-1-HA receptor and the mCherry reporter induces a co-translational self-cleavage, leaving behind the receptor-HA tag fusion and the cytosolic mCherry tag. The activation of gene expression was induced by illumination with 660 nm for the red light system (Fig. 1) and 465 nm for the blue light system (Figs. 2, 3). When the cells containing the light inducible receptor were not exposed to light (dark controls), no mCherry was detected in the cytosol, i.e. no receptor was expressed and transported to the cell surface.

Since our viral vector was not able to integrate into cells not expressing mCAT-1 on their surface, it was not able to infect non-illuminated cells (Figs. 2, 3). As a reporter to detect the cells that were infected by viral particles, we used EGFP as a cargo. In principle any gene of interest could be transduced under light control using The AcCELLerator.

We determined the expression time of the receptor that was induced by blue light after illumination for five hours. We used a receptor that was labeled with a fused HA-tag and mCherry separated by a P2A site (p14ls_003), for analysis with Western blot and fluorescence microscopy. The receptor had an expression peak at 24 hours after beginning of illumination.

Pattern Generation

For generating pattern in homogeneous cell layers, we transfected HEK293T cells with the blue light system and the blue light induced receptor (p14ls_003). Dishes were covered with a photo mask preventing areas in the cell culture from light exposure. However, the blue light system is very sensitive to even low intensities of blue light. Due to scatterd light, the receptor was activated in a large area of the cell culture. Thus patterns were not visible at this time point due to activation of receptor expression by scattered light (Fig. 5). Therefore, we also did not apply the virus to these cultures.