Team:Freiburg/Content/Results/Vector
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<p>An important aspect for the function of our system as well as for its safety is the specificity of the vector regarding infection of different | <p>An important aspect for the function of our system as well as for its safety is the specificity of the vector regarding infection of different | ||
kind of cells. The vector deriving from the murine leukemia virus is specific for cells carrying the mouse specific CAT-1. Cells that do not have | kind of cells. The vector deriving from the murine leukemia virus is specific for cells carrying the mouse specific CAT-1. Cells that do not have | ||
this specific receptor are not targeted by the vector. In order to test the specificity of the system, different kind of cells were incubated with | this specific receptor are not targeted by the vector. In order to test the specificity of the system, different kind of cells were incubated with | ||
the vector containing EGFP. Since EGFP is stable integrated by the system, infected cells are identified by a green fluorescence that was analysed | the vector containing EGFP. Since EGFP is stable integrated by the system, infected cells are identified by a green fluorescence that was analysed | ||
- | via flow cytometry (figure 1). We tested two human cell lines, human embryonic kidney cells as well as human lung epithel carcinoma cells, for their | + | via flow cytometry (figure 1). |
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+ | <p>We tested two human cell lines, human embryonic kidney cells as well as human lung epithel carcinoma cells, for their | ||
capability of being targeted by the vector. In addition, mouse fibroblasts that express the mouse specific CAT-1 receptor were tested for a positive | capability of being targeted by the vector. In addition, mouse fibroblasts that express the mouse specific CAT-1 receptor were tested for a positive | ||
control. As shown in figure 1 both human cell lines did not express EGFP after incubation with the vector indicating that they were not targeted. However, | control. As shown in figure 1 both human cell lines did not express EGFP after incubation with the vector indicating that they were not targeted. However, | ||
many cells of the mouse cell line were expressing EGFP after infection. | many cells of the mouse cell line were expressing EGFP after infection. | ||
</p> | </p> | ||
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<img src="https://static.igem.org/mediawiki/2014/f/ff/Freiburg2014-10-02_results_skizze_specificity.JPG"> | <img src="https://static.igem.org/mediawiki/2014/f/ff/Freiburg2014-10-02_results_skizze_specificity.JPG"> | ||
<figcaption> | <figcaption> | ||
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<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/2014/4/45/Freiburg2014-09-10_FACS-specificity-HEK-MOUSE.png"> | <img src="https://static.igem.org/mediawiki/2014/4/45/Freiburg2014-09-10_FACS-specificity-HEK-MOUSE.png"> | ||
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</figcaption> | </figcaption> | ||
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<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/2014/5/5a/Freiburg2014-09-10_cell-line-specificity.png"> | <img src="https://static.igem.org/mediawiki/2014/5/5a/Freiburg2014-09-10_cell-line-specificity.png"> | ||
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</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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<blockquote><strong>The vector derived from the murine leukemia virus is specific for the murine CAT-1 receptor. Therefore | <blockquote><strong>The vector derived from the murine leukemia virus is specific for the murine CAT-1 receptor. Therefore | ||
it is not able to infect human cell lines!</blockquote> | it is not able to infect human cell lines!</blockquote> |
Revision as of 15:03, 2 October 2014
1 The Vector
1.1 Specificity of MuLV
An important aspect for the function of our system as well as for its safety is the specificity of the vector regarding infection of different kind of cells. The vector deriving from the murine leukemia virus is specific for cells carrying the mouse specific CAT-1. Cells that do not have this specific receptor are not targeted by the vector. In order to test the specificity of the system, different kind of cells were incubated with the vector containing EGFP. Since EGFP is stable integrated by the system, infected cells are identified by a green fluorescence that was analysed via flow cytometry (figure 1).
We tested two human cell lines, human embryonic kidney cells as well as human lung epithel carcinoma cells, for their capability of being targeted by the vector. In addition, mouse fibroblasts that express the mouse specific CAT-1 receptor were tested for a positive control. As shown in figure 1 both human cell lines did not express EGFP after incubation with the vector indicating that they were not targeted. However, many cells of the mouse cell line were expressing EGFP after infection.
The vector derived from the murine leukemia virus is specific for the murine CAT-1 receptor. Therefore it is not able to infect human cell lines!
1.2 Optimization of transduction