"
Team:Freiburg/Content/Results/Vector
From 2014.igem.org
Mirja Harms (Talk | contribs) |
Mirja Harms (Talk | contribs) |
||
| Line 14: | Line 14: | ||
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..</p> | + | 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 | ||
| + | capaticity 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. | ||
| + | </p> | ||
</div> | </div> | ||
<div class="col-sm-6"> | <div class="col-sm-6"> | ||
| Line 20: | Line 25: | ||
<img src="https://static.igem.org/mediawiki/2014/f/fd/Freiburg2014-09-10_vector-specificity_pic1.1.png"> | <img src="https://static.igem.org/mediawiki/2014/f/fd/Freiburg2014-09-10_vector-specificity_pic1.1.png"> | ||
<figcaption> | <figcaption> | ||
| - | <p class="header">Fig. | + | <p class="header">Fig.1: Scheme for testing the specificity of the vector.</p> |
<p class="desc">In order to test the specificity of the viral vector different cells were incubated with the vector for four hours. Cells not containing the mouse specific receptor | <p class="desc">In order to test the specificity of the viral vector different cells were incubated with the vector for four hours. Cells not containing the mouse specific receptor | ||
CAT-1 are not capable for infection. As control mouse fibroblasts (NIH3t3) expressing CAT-1 were used.</p> | CAT-1 are not capable for infection. As control mouse fibroblasts (NIH3t3) expressing CAT-1 were used.</p> | ||
| Line 38: | Line 43: | ||
</figure> | </figure> | ||
</section> | </section> | ||
| + | |||
| + | |||
<h2>1.2 Optimization of transduction</h2> | <h2>1.2 Optimization of transduction</h2> | ||
Revision as of 23:03, 1 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 capaticity 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.
Fig.1: Scheme for testing the specificity of the vector.
In order to test the specificity of the viral vector different cells were incubated with the vector for four hours. Cells not containing the mouse specific receptor CAT-1 are not capable for infection. As control mouse fibroblasts (NIH3t3) expressing CAT-1 were used.
Fig.X: FACS analysis of different cell lines incubated with the CAT-1 specific viral vector
Left: Different cell lines not incubated with the vector as negative control, Middle: different cell lines infected with MuLV IRES EGFP (50% in completed growth medium), Right: cells transfected with the mouse specific receptor CAT-1 (rz006) were infected with the viral vector. The infection efficiency is directly correlated with transfection efficiency of the receptor into the different kind of cells.
