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Team:Freiburg/Content/Results/Light system
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<h1>The light system</h1> | <h1>The light system</h1> | ||
| - | <p>One advantage of our system, the | + | <p>One advantage of our system, the spatial resolution by infecting only distinct areas in a mammalian cell culture with our viral vector, was archived by the use of different light systems. We induced the expression of the mCAT-1 receptor in non-murine cell lines by illumination them with light of a special wave length. Since, the viral vector only infect cells that express the mCAT-1 receptor on their surfaces, we transferred genes into distinct areas of an otherwise homogenous cell layer using this principle. |
</p> | </p> | ||
<h2>Function of the light systems</h2> | <h2>Function of the light systems</h2> | ||
| - | <p> As the function of the light system, we use, is really important for the function of our whole system, we tested two different light systems. The red light system is based on…. whereas the blue light system | + | <p> As the function of the light system, we use, is really important for the function of our whole system, we tested two different light systems. The red light system is based on…. whereas the blue light system uses… blubb |
</p> | </p> | ||
| - | <p>Since, it is known that the red light system works better in chinese hamster ovary (CHO) cells, we used | + | <p>Since, it is known that the red light system works better in chinese hamster ovary (CHO) cells, we used this cell line to test the function of this light system. In order to investigate the functionality of the blue light system, we tested both, CHO cells and human embryonic kidney (HEK293) cells. As reporter the gene seap was transfected together with the genes for the light systems (red light system: PKM006, PKM078; blue light system: PKM292, PKM297, PKM084). A SEAP-assay was performed after illumination. Regarding SEAP concentrations in the supernatant of each sample two important findings were archived. First, the blue light system works much more efficient than the red light system; and the blue light system works better with HEK cells than with CHO cells. |
</p> | </p> | ||
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</figure> | </figure> | ||
| - | <p>The blue light system is very effective in activating an existing reporter brought into the cell before illumination. As we know that the duration of illuminating the system with blue light (452 nm) is critical for its efficiency, we tested various time spans for illumination. Our results indicate that five hours of illumination lead to the | + | <p>The blue light system is very effective in activating an existing reporter, like the gene for seap, brought into the cell before illumination. As we know that the duration of illuminating the system with blue light (452 nm) is critical for its efficiency, we tested various time spans for illumination. Our results indicate that five hours of illumination lead to the highest activation level of SEAP expression. |
</p> | </p> | ||
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</figure> | </figure> | ||
| - | <p>Another important finding was the specificity of the blue light system. As we always have a dark control in our experiments, we | + | <p>Another important finding was the specificity of the blue light system. As we always have a dark control in our experiments, we determined almost no activation of the SEAP reporter in these samples leading to a very low background level in our system. |
</p> | </p> | ||
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</p> | </p> | ||
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| + | <p> | ||
| + | </p> | ||
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<h2>SEAP as reporter</h2> | <h2>SEAP as reporter</h2> | ||
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<h2>MuLV SEAP</h2> | <h2>MuLV SEAP</h2> | ||
Revision as of 12:04, 15 October 2014
The light system
One advantage of our system, the spatial resolution by infecting only distinct areas in a mammalian cell culture with our viral vector, was archived by the use of different light systems. We induced the expression of the mCAT-1 receptor in non-murine cell lines by illumination them with light of a special wave length. Since, the viral vector only infect cells that express the mCAT-1 receptor on their surfaces, we transferred genes into distinct areas of an otherwise homogenous cell layer using this principle.
Function of the light systems
As the function of the light system, we use, is really important for the function of our whole system, we tested two different light systems. The red light system is based on…. whereas the blue light system uses… blubb
Since, it is known that the red light system works better in chinese hamster ovary (CHO) cells, we used this cell line to test the function of this light system. In order to investigate the functionality of the blue light system, we tested both, CHO cells and human embryonic kidney (HEK293) cells. As reporter the gene seap was transfected together with the genes for the light systems (red light system: PKM006, PKM078; blue light system: PKM292, PKM297, PKM084). A SEAP-assay was performed after illumination. Regarding SEAP concentrations in the supernatant of each sample two important findings were archived. First, the blue light system works much more efficient than the red light system; and the blue light system works better with HEK cells than with CHO cells.
Test
The blue light system is very effective in activating an existing reporter, like the gene for seap, brought into the cell before illumination. As we know that the duration of illuminating the system with blue light (452 nm) is critical for its efficiency, we tested various time spans for illumination. Our results indicate that five hours of illumination lead to the highest activation level of SEAP expression.
Test
Another important finding was the specificity of the blue light system. As we always have a dark control in our experiments, we determined almost no activation of the SEAP reporter in these samples leading to a very low background level in our system.
Light induced receptor/ blue light
In order to generate viral induced patterns in a homogenous cell culture, we combined the spatial resolution of the light system with the specificity of viral receptor interactions. Since our viral vector is specific for the mCAT-1 receptor that is under normal conditions only expressed in murine cells, we linked this specific receptor to the blue light system and transfected non-murine cells with this receptor combination. Mammalian cells which contain the inducible receptor expressed mCAT-1 after illumination and were infected with our viral vector leading to EGFP expression. Cells of the same cell culture that were not illuminated were not able to express the receptor leading to no infection with our viral vector.
