Team:Freiburg/Content/Results/Vector

From 2014.igem.org

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<h2>1.1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Specificity of MuLV</h2>
<h2>1.1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Specificity of MuLV</h2>
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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  
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          <img src="https://static.igem.org/mediawiki/2014/f/ff/Freiburg2014-10-02_results_skizze_specificity.JPG">
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            <p class="header">Fig.1: Description of the experiment (as picture)</p>
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<p class="header">Fig.1: Description of the experiment (as picture)</p>
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        <p class="header">Fig.X: FACS analysis of different cell lines incubated with the CAT-1 specific viral vector</p>
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        <p class="desc">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  
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<p class="header">Fig.X: FACS analysis of different cell lines incubated with the CAT-1 specific viral vector</p>
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transfected with the mouse specific receptor CAT-1 (rz006) were infected with the viral vector. The infection efficiency is directly correlated with transfection  
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<p class="desc">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  
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efficiency of the receptor into the different kind of cells.</p>
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transfected with the mouse specific receptor CAT-1 (rz006) were infected with the viral vector. The infection efficiency is directly correlated with transfection  
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efficiency of the receptor into the different kind of cells.</p>
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            <p class="header">Fig.1: Infection of different cell lines with the viral vector derived from the murine leukemia virus.</p>
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<p class="header">Fig.1: Infection of different cell lines with the viral vector derived from the murine leukemia virus.</p>
<p class="desc">In order to investigate the specificity of the viral vector, two human cell lines, human embryonic kidney cells as well as lung  
<p class="desc">In order to investigate the specificity of the viral vector, two human cell lines, human embryonic kidney cells as well as lung  
epithel carcinoma cells, were infected and the percentage of cells expressing EGFP was analyzed by flow cytometry.</p>
epithel carcinoma cells, were infected and the percentage of cells expressing EGFP was analyzed by flow cytometry.</p>
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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  
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<h2>1.2&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Optimization of transduction</h2>
<h2>1.2&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Optimization of transduction</h2>
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<p class="header">Fig.: Transduction efficiency of the viral vector in mouse cells.</p>
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<p class="header">Fig.: Transduction efficiency of the viral vector in mouse cells.</p>
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<h2>1.3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Expression of different viral constructs</h2>
<h2>1.3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Expression of different viral constructs</h2>

Revision as of 14:51, 2 October 2014

The AcCELLerator

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.

Fig.1: Description of the experiment (as picture)

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.

Fig.1: Infection of different cell lines with the viral vector derived from the murine leukemia virus.

In order to investigate the specificity of the viral vector, two human cell lines, human embryonic kidney cells as well as lung epithel carcinoma cells, were infected and the percentage of cells expressing EGFP was analyzed by flow cytometry.

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

figure>

Fig.: Transduction efficiency of the viral vector in mouse cells.

Fig.: Transduction efficiency of the viral vector in mouse cells.

1.3         Expression of different viral constructs

1.4         Virus dilution and concentration

1.5         Half life time of MuLV

1.6         Integration time of MuLV

1.7         Stable integration

1.8         Stable Cell line with MuLV

2          The Receptor

2.1         Localisation Receptor

2.2         Expression of Receptor using different DNA concentrations

2.3         Receptor expression time

2.4         Receptor life time after splitting

3          The light system

3.1         Function of blue light system

3.2         Function of red light system

3.3         Light induced receptor/ blue light

3.4         Receptor functionality

3.5         SEAP as reporter

3.6         MuLV SEAP

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