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Team:Freiburg/Content/Results/Receptor
From 2014.igem.org
The Receptor
The specificity of our system is based on the murine CAT-1 receptor which serves as the viral entry site of our vector on target cells. We manage not only to stably transfer genes into the genomes of cells, we also managed to target specific cells in a tissue by inducing the expression of the murine CAT-1 receptor in these cells we want to target. Since, mCAT-1 is naturally only present in murine cell lines, we use human cell lines for transfecting them with the receptor gene. Results indicate that human cell lines that express the mCAT-1 receptor, are specific targeted by our viral particles, thus, making particular gene transfer possible.
Localisation Receptor
Since, the mCAT-1 receptor serves as the entry site of our viral vector, it is essential that it is expressed on the surface of target cells. We managed to express the murine CAT-1 receptor in human cell lines. In order to determinate the location of our receptor, we labeled it with the fluorescence marker mCherry and transferred it into human embryonic kidney (HEK) cells. Confocal microscopy of cells, expressing this labeled receptor, indicates its location on the surface of the cells.
Receptor Functionality
We showed that we managed to transduce genes with a high efficiency into murine cell lines which naturally express the mCAT-1 receptor. Regarding our system we want to express the mCAT-1 receptor on human cell lines. Therefore it was of supreme importance to test the functionality of the transient expressed mCAT-1 in these cells. As we know that the receptor is expressed on the surface of human cells, if transfected with receptor DNA, we tested the function of mCAT-1 by infecting them with our vector containing EGFP as a marker. Results indicate that the receptor is not only expressed in different non-murine cell lines but can be also used as target for viral infection.
Receptor expression time
The exact time point for viral infection was adjusted to the time the receptor needs for expression in target cells. In order to determine the expression time of mCAT-1 in HEK cells, the receptor was labeled with an HA-tag making antibody detection possible. Cells transfected with receptor DNA were analyzed regarding different durations of incubation. Results indicate that our receptor is highly expressed after 24 hours of incubation denoting that the perfect time point for viral infection is in the same range.
