Team:Freiburg/Content/Results/Receptor

From 2014.igem.org

(Difference between revisions)
 
(22 intermediate revisions not shown)
Line 6: Line 6:
<body>
<body>
<section id="Receptor">
<section id="Receptor">
 +
<div class="row category-row">
 +
<div class="col-sm-6">
 +
<div class="container-fluid" style="float: left">
 +
<div style="position: relative; float: right; margin-top: 4px;">
 +
<a href="https://2014.igem.org/Team:Freiburg/Results/Vector">Go back to The Vector</div>
 +
<div style="position: relative; float: left;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/4/44/Freiburg2014_Navigation_Arrow_rv.png">  <!-- Pfeil rv--></a></div>
 +
</div>
 +
</div>
 +
<div class="col-sm-6">
 +
<div class="container-fluid" style="float: right">
 +
<div style="position: relative; float: left; margin-top: 4px;">
 +
<a href="https://2014.igem.org/Team:Freiburg/Results/Light_system">See the Results of our Ligth Systems</div>
 +
<div style="position: relative; float: right;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/9/95/Freibur2014_pfeilrechts.png">  <!-- Pfeil fw--></a></div>
 +
</div>
 +
</div>
 +
</div>
-
<h1>The Receptor</h1>
+
<h1>The Receptor</h1>
-
<p>The specificity of our system is based on the murine CAT-1 receptor, which serves as the viral entry site of our vector into target cells. We could show that our viral vector system can be used to stably integrate genes into the genome of murine cells. We could also target any other cell line by transferring the gene for the murine CAT-1 receptor in these cells before transduction. Since mCAT-1 is naturally only present in murine cell lines, we use human cell lines for transfection of the receptor gene. Expression of a reporter demonstrates that only the cells expressing the mCAT-1 receptor are infected by the viral particles, thus making particular gene transfer possible.
+
<p>
-
</p>
+
  The specificity of our system is based on the murine CAT-1 receptor, which serves
 +
  as the viral entry site of our viral vector into the target cells. We demonstrate
 +
  that the virus can be used to stably integrate genes into the genome of murine cells.
 +
  We also targeted other cell lines by expressing the gene for the murine CAT-1 receptor
 +
  in these cells before viral transduction. Since mCAT-1 is naturally only present in murine
 +
  cell lines, we use human cell lines for transfection of the receptor. Expression of
 +
  a reporter suggests that only the cells expressing the mCAT-1 receptor are being
 +
  infected by the viral particles, thus making particular gene transfer possible.
 +
</p>
 +
<h2 id="Results-Receptor-LocalisationReceptor">Subcellular Localization of the Receptor</h2>
-
+
<p>
-
<h2 id="Results-Receptor-LocalisationReceptor">Localisation Receptor</h2>
+
  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. In order to determine the localization
 +
  of the mCAT-1 receptor, we labeled the C-terminus of the mCAT-1 with the fluorescent protein
 +
  mCherry and transfected it into human embryonic kidney (HEK-293T) cells. After distinct
 +
  time points cells were imaged with a confocal scanning laser microscope. The mCAT1-mCherry
 +
  construct was found predominantly at the surface of the cells (Figs. 1, 2).
 +
</p>
 +
 
 +
<div class="row category-row">
 +
  <div class="col-sm-6">
 +
    <figure class="fig-full-width">
 +
      <a href="https://static.igem.org/mediawiki/2014/d/d0/Freiburg2014_Receptor_localization_01_overview.png">
 +
        <img class="img-no-pad"
 +
          src="https://static.igem.org/mediawiki/2014/d/d0/Freiburg2014_Receptor_localization_01_overview.png">
 +
      </a>
 +
      <figcaption>
 +
        <p class="header">
 +
          Figure 1: Overview image of HEK-293T cells transfected with mCAT-1-mCherry (p14rz_005).
 +
        </p>
 +
        <p class="desc">
 +
          Confocal pictures were taken with a 20x plan apo objective. Nuclear staining (DAPI) is
 +
          shown in blue  and the mCAT-1-mCherry in red.<a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#MirjaHarms_Localization">Labjournal</a></p>
 +
        </p>
 +
      </figcaption>
 +
    </div>
 +
    <div class="col-sm-6">
 +
      <figure class="fig-full-width">
 +
        <a href="https://static.igem.org/mediawiki/2014/0/00/Freiburg2014_Receptor_localization_04.png">
 +
          <img class="img-no-pad"
 +
            src="https://static.igem.org/mediawiki/2014/0/00/Freiburg2014_Receptor_localization_04.png">
 +
        </a>
 +
      <figcaption>
 +
        <p class="header">
 +
          Figure 2: HEK-293T cells transfected with mCAT-1-mCherry (p14rz_005).
 +
        </p>
 +
        <p class="desc">
 +
          Confocal pictures were taken with an 100x oil immersion objective (NA 1.40).
 +
          Nuclear staining (DAPI) is shown in blue and the mCAT-1-mCherry in red.
 +
        </p>
 +
      </figcaption>
 +
    </figure>
 +
  </div>
 +
</div>
-
<p>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. In order to determinate the localization of the mCAT-1 receptor, we labeled the C-terminus of the mCAT-1 with the fluorescence protein mCherry and transfected this construct in human embryonic kidney (HEK-293T) cells. After distinct time points cells were imaged with a confocal scanning laser microscope. The mCAT1-mCherry was found predominantly at the surface of the cells (Fig. 1-2).
+
<h2 id="Results-Receptor-ReceptorFunctionality">Receptor Functionality and Virus Specificity</h2>
-
</p>
+
-
<div class="row category-row">
+
<p>
-
<div class="col-sm-6">
+
  Transduction of genes into murine cell lines that naturally express the mCAT-1
-
+
  receptor occurred with an efficiency of over 80%. For our system we needed functional
-
<figure class="fig-full-width">
+
  expression of the receptor in non-murine cell lines, i.e. the receptor has to serve
-
<a href="https://static.igem.org/mediawiki/2014/d/d0/Freiburg2014_Receptor_localization_01_overview.png"> <!-- ORGINAL -->
+
  as an entry site for the virus, and the virus has to efficiently deliver its cargo
-
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/d/d0/Freiburg2014_Receptor_localization_01_overview.png"> <!-- Thumbnail -->
+
  into the target cell. We tested the functionality of the mCAT-1 receptor by expressing
-
</a>
+
  it in HEK-293T cells and subsequent infection with the virus containing EGFP as a cargo.
-
<figcaption>
+
  The presence of green fluorescent cells in the infected cultures of different non-murine
-
<p class="header">Figure 1: Overview image of HEK293T cells transfected with mCAT-1-mCherry (p14xxx) taken with 20x plan apo objective. Nuclear staining is shown in blue (DAPI) and in red the mCAT-1-mCherry.</p>
+
  cell lines indicated that the receptor was not only expressed but can also be used
-
</figcaption>
+
  as an entry site by the virus.
-
</div>
+
</p>
-
<div class="col-sm-6">
+
<p>
-
+
  During our project we generated receptor constructs which were labeled with fluorescent
-
<figure class="fig-full-width">
+
  protein (for microscopy) or the HA tag (for Western blotting). Since we didn't know
-
<a href="https://static.igem.org/mediawiki/2014/0/00/Freiburg2014_Receptor_localization_04.png"> <!-- ORGINAL -->
+
  whether the tags have an impact on viral infection capabilities, we tested them for
-
<img src="https://static.igem.org/mediawiki/2014/0/00/Freiburg2014_Receptor_localization_04.png"> <!-- Thumbnail -->
+
  their functionality, confirming that all receptor constructs lead to viral infection
-
</a>
+
  when expressed in non-murine cell lines (Fig. 3). For making the receptor visible for
-
<figcaption>
+
  fluorescent microscopy and analysis by flow cytometry, we used the mCherry tag
-
<p class="header">Figure 2: HEK293 cells transfected with mCAT-1-mCherry (p14rz_005) taken with 100x oil immersion objective 1.4 NA. Nuclear staining is shown in blue (DAPI) and in red the mCAT-1-mCherry.</p>
+
  (constructs p14rz_005 and p14rz_006).  
-
</figcaption>
+
</p>
-
+
 
-
        </figure>
+
<figure class="fig-full-width">
-
</div>
+
  <a href="https://static.igem.org/mediawiki/2014/9/9d/Freiburg2014_Results_HEK_verschiedene_receptoren.jpg">
-
</div>
+
    <img class="img-no-pad"
-
+
      src="https://static.igem.org/mediawiki/2014/9/9d/Freiburg2014_Results_HEK_verschiedene_receptoren.jpg">
-
<h2 id="Results-Receptor-ReceptorFunctionality">Receptor Functionality</h2>
+
  </a>
-
+
  <figcaption>
-
<div class="row category-row">
+
    <p class="header">
-
<div class="col-sm-6">
+
      Figure 3: HEK-293T cells transfected with different receptor constructs
-
<p>Transduction of genes into murine cell lines which naturally express the mCAT-1 receptor occurred with an efficiency over 80%. For our system we need a functional expression of the receptor in non-murine cell lines, i.e. the receptor has to serve as an entry site for the virus, and the virus has to efficiently deliver its cargo into the target cell. We tested the functionality of the mCAT-1 receptor by infecting HEK-293T cells expressing the receptor with the virus containing eGFP as a cargo. The presence of green fluorescent cells in the infected cultures of different non-murine cell lines indicates that the receptor is not only expressed but can be also used as an entry site by the virus.
+
      and infected with MuLV-EGFP afterwards.
-
</p>
+
    </p>
-
<p>During our projekt we generated different receptor constructs all of them labeled with different markers. Since we didn't know, if these tags have an influence on viral infection capabilities, we tested all of them for their functionality determining that all receptor constructs lead to viral infection if expressed in non murine cell lines (Fig.4-5). For making the receptor visible for fluorescent microscopy and analysis by flow cytometry, we labelled the receptor with mCherry (p14rz_005 and p14rz_006). Analysis of cells expressing mCAT-1-mCherry that were infected with MuLV EGFP showed that all cells which were transduced by the viral particles also express the receptor, i.e. only cells expressing the receptor were infected (Fig. 3). 
+
    <p class="desc">
-
</p>
+
      Cells transfected with (A) mCAT-1, (B) mCAT-1-HA and (C) mCAT-1-HA-mCherry (red).
-
+
      Cells were transduced with MuLV-EGFP after 24 h. Pictures were taken after 48 h.
-
</div>
+
      EGFP fluorescence is shown in green. <a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#MirjaHarms_differnt_kinds_of_receptors">Labjournal</a>
-
<div class="col-sm-6">
+
    </p>
-
<figure>
+
  </figcaption>
-
<a href="https://static.igem.org/mediawiki/2014/2/25/Freiburg2014_Results_HEK_prz006_mulv_together.jpg"> <!-- ORGINAL -->
+
</figure>
-
<img src="https://static.igem.org/mediawiki/2014/2/25/Freiburg2014_Results_HEK_prz006_mulv_together.jpg"> <!-- Thumbnail -->
+
-
</a>
+
-
<figcaption>
+
-
<p class="header">Figure 3: FACS data of HEK-293T cells tranfected with the receptor and transduced with MuLV eGFP.</p>
+
-
<p class="desc">HEK-293T cells were transfected with the p14rz_006 which was labeled with mCherry and infected with MuLV transferring eGFP. Cells were analyzed with flow cytometry 48 h after transduction.</p>
+
-
</figcaption>
+
-
        </figure>
+
-
 
-
</div>
 
-
</div>
 
-
 
-
<figure class="fig-full-width">
 
-
            <a href="https://static.igem.org/mediawiki/2014/9/9d/Freiburg2014_Results_HEK_verschiedene_receptoren.jpg"> <!-- ORGINAL -->
 
-
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/9/9d/Freiburg2014_Results_HEK_verschiedene_receptoren.jpg"> <!-- Thumbnail -->
 
-
            </a>
 
-
<figcaption>
 
-
<p class="header">Figure 4: HEK-293T cells transfected with different receptor constructs and infected with MuLV eGFP afterwards.</p>
 
-
<p class="desc">The cells were transfected with pQCXIN (original vector containing SLC7A1) (A), p14rz_004 (labeled with HA-tag)(B) and p14rz_006 (labeled with HA-tag and mCherry)(C). Cells were transduced with MuLV after 24 h. Pictures were taken after 48 h.</p>
 
-
</figcaption>
 
-
</figure>
 
-
 
-
<div class="row category-row">
 
-
<div class="col-sm-6">
 
-
 
-
<figure>
 
-
            <a href="https://static.igem.org/mediawiki/2014/b/b0/Freiburg2014_Receptor_08_plus_Virus_single_channels.png"> <!-- ORGINAL -->
 
-
<img class="img-no-pad" src="https://static.igem.org/mediawiki/2014/b/b0/Freiburg2014_Receptor_08_plus_Virus_single_channels.png"> <!-- Thumbnail -->
 
-
            </a>
 
-
<figcaption>
 
-
<p class="header">Figure 4: HEK293T cells expressing mCAT-1-mCherry transduced with MuLV-GFP.</p>
 
-
<p class="desc">Overlay of all three channels (A); DAPI stained nuclei (B); EGFP expression in infected cells (C); mCAT-1-mCherry expression on the surface of HEK293T cells (D) Objective plan apo 60x, 1.4 NA.</p>
 
-
</figcaption>
 
-
</figure>
 
-
</div>
 
-
<div class="col-sm-6">
+
<div class="row category-row">
-
+
  <div class="col-sm-6">
-
<p>
+
    <p>
-
Regarding the expression rate of the receptor on the surface of HEK293T cells, we noticed that mCAT-1-mCherry is less present after transduction with our viral particles. That means that the receptor is eather internalized after infection by a mechanism initiated by the virus, or the expression rate of the receptor decreases after infection. Since the particles can only transfer their cargo into dividing cells we found often pairs of cells that were infected by the viral vector or at least high expression rate of the receptor in cells next to targeted cells.
+
      Owing to the limited resolution and sensitivity of the cell culture microscope used
-
</p>
+
      for the images in Fig. 3, it was not clear whether the cells expressing the receptor
-
+
      were the same cells that were transduced by the virus and expressed EGFP (Fig. 3C).
-
</div>
+
      Therefore we obtained higher qualtiy images with a confocal microscope (Fig. 4). The
-
</div>
+
      cells were transfected with the mCAT-1-mCherry construct, 24h later infected by
-
+
      the virus containing EGFP, and imaged 48h later. We found many cells that expressed
-
<h2 id="Results-Receptor-ReceptorExpression">Receptor Expression</h2>
+
      EGFP in the cytoplasm and the mCAT-1 receptor on the plasma membrane, whereas we did not
 +
      find cells that expressed EGFP but not the receptor, suggesting that
 +
      mCAT-1 expression is required for infection of the cells by the MuLV.
 +
    </p>
 +
    <p>
 +
      We also noticed that mCAT-1-mCherry was less present after transduction with
 +
      the viral particles. Possible explanations are that the receptor is internalized
 +
      after infection by a mechanism initiated by the virus, or that the expression
 +
      rate of the receptor decreases after infection. Since the particles can only
 +
      transfer their cargo into dividing cells, we often found pairs of cells that were
 +
      infected by the viral vector or observed high expression of the receptor in
 +
      cells next to targeted cells.
 +
    </p>
 +
    <p>
 +
      We were wondering whether receptor expression is indeed required for virus entry into the cell.
 +
      Although we observed by confocal microscopy that many cells that expressed EGFP
 +
      also expressed the receptor, we set out to correlate the expression levels of receptor and
 +
      EGFP, i.e. the cargo of the virus. Therefore we used FACS analysis to quantify
 +
      the levels of mCherry and EGFP fluorescence in all cells (Fig. 5). Although many cells that expressed
 +
      the receptor were not transduced with EGFP, we found that all cells that expressed EGFP
 +
      also expressed the receptor (Fig. 5, top right quadrant). Only a negligible number
 +
      of cells showed EGFP expression but low mCherry fluorescence (Fig. 5, top left quadrant).
 +
      This requirement for the presence of the receptor to allow efficient virus entry indicated
 +
      that the MuLV is indeed highly specific for the mCAT-1 receptor.
 +
    </p>
 +
<video src="https://static.igem.org/mediawiki/2014/6/6b/Freiburg2014_confocal_HEK293T_mCAT1.mp4" type="video/mp4" width="480" height="360" autobuffer autoplay loop>
 +
    </video>
 +
  </div>
 +
  <div class="col-sm-6">
 +
    <figure>
 +
      <a href="https://static.igem.org/mediawiki/2014/b/b0/Freiburg2014_Receptor_08_plus_Virus_single_channels.png">
 +
        <img class="img-no-pad"
 +
          src="https://static.igem.org/mediawiki/2014/b/b0/Freiburg2014_Receptor_08_plus_Virus_single_channels.png">
 +
      </a>
 +
      <figcaption>
 +
        <p class="header">
 +
          Figure 4: HEK-293T cells expressing mCAT-1-mCherry transduced with MuLV-GFP.
 +
        </p>
 +
        <p class="desc">
 +
          (A) Overlay of all three channels, (B) DAPI, (C) EGFP and (D) mCAT-1-mCherry.
 +
          Objective plan apo 60x, NA 1.40. <a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#MirjaHarms_Localization_receptor_Virus">Labjournal</a>
 +
        </p>
 +
      </figcaption>
 +
    </figure>
 +
 
 +
    <figure>
 +
      <a href="https://static.igem.org/mediawiki/2014/2/25/Freiburg2014_Results_HEK_prz006_mulv_together.jpg">
 +
        <img src="https://static.igem.org/mediawiki/2014/2/25/Freiburg2014_Results_HEK_prz006_mulv_together.jpg">
 +
      </a>
 +
      <figcaption>
 +
        <p class="header">
 +
          Figure 5: FACS data of HEK-293T cells tranfected with the receptor and
 +
          infected with MuLV-EGFP.
 +
        </p>
 +
<p class="desc">
 +
          HEK-293T cells were transfected with the mCAT-1-mCherry and infected with MuLV-EGFP.
 +
          Cells were analyzed by flow cytometry 48 h after infection. <a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#MirjaHarms_differnt_kinds_of_receptors">Labjournal</a>
 +
        </p>
 +
      </figcaption>
 +
    </figure>
 +
  </div>
 +
</div>
 +
 
 +
 
 +
 
 +
<h2 id="Results-Receptor-ReceptorExpression">Receptor Expression</h2>
-
<p>The 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-293T cells, we transfected the cells with the HA-tagged mCAT-1 (p14rz_006). Cells transfected with receptor DNA were analyzed after different incubation times. As the expression of the receptor peaks at 24 h after transfection, we used this time point for viral infections (Fig. 6).  
+
<p>
-
</p>
+
To enable efficient infection of cells with the virus, the time point for viral infection should match the time the receptor needs for expression in target cells. In order to determine the expression time of mCAT-1 in HEK-293T cells, we transfected the cells with the HA-tagged mCAT-1. Cells transfected with receptor DNA were analyzed after different incubation times. We found that the expression of the receptor peaked at 24 h after transfection (Fig. 6). In later experiments we used this time point for viral infections.
 +
</p>
 +
<p>
 +
Since the presence of mCAT-1 on the cell surface is a main factor for viral tranduction efficiency, we quantified the expression of the receptor after transfection of HEK-293T cells with different concentrations of receptor DNA by Western blotting. We found that the expression of the receptor in the cell increases with higher amounts of transfected DNA (Fig. 7). However, we did not use such high DNA concentrations for our later experiments because we found that non-murine cells transfected with high amounts of transfected DNA died, probably due to overexpression of the mCAT-1 receptor. 
 +
</p>
-
<div class="row category-row">
+
<div class="row category-row">
-
<div class="col-sm-6">
+
  <div class="col-sm-6">
-
<figure>
+
    <figure>
-
<img src="https://static.igem.org/mediawiki/2014/5/5b/Freiburg2014_Results_WB_receptorexpressiontime.jpg">
+
      <img src="https://static.igem.org/mediawiki/2014/5/5b/Freiburg2014_Results_WB_receptorexpressiontime.jpg">
-
<figcaption>
+
      <figcaption>
-
<p class="header">Figure 6: Expression time of the receptor that was transfected into HEK293 cells.</p>
+
        <p class="header">
-
<p class="desc">After transfection with p14rz_006 (HA-labeled) cells were lysed with RIPA buffer at distinct time points. A Western blot was performed using anti-HA antibody.</p>
+
          Figure 6: Expression time of the receptor that was transfected into HEK-293T cells.
-
</figcaption>
+
        </p>
-
</figure>
+
        <p class="desc">
-
</div>
+
          After transfection with mCAT-1-HA cells were lysed with RIPA buffer  
-
+
          at distinct time points. A Western blot was performed using an anti-HA antibody. <a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#MirjaHarms_Expression_time_receptor">Labjournal</a>
-
<div class="col-sm-6">
+
        </p>
-
<figure>
+
      </figcaption>
-
<img src="https://static.igem.org/mediawiki/2014/c/cc/Freiburg2014_Results_WB_receptorexpression_DNA_conc.jpg">
+
    </figure>
-
<figcaption>
+
  </div>
-
<p class="header">Figure 7: Tranfection of HEK-293T cells with different receptor DNA concentrations.</p>
+
  <div class="col-sm-6">
-
<p class="desc">Cells (on 35mm plates) were transfected with 0,6 µg to 5,4 µg p14rz_006 (HA-labeled) per well. Cells were lysed with RIPA buffer after 24 h of incubation and analysed by Western blotting.</p>
+
    <figure>
-
</figcaption>
+
      <img src="https://static.igem.org/mediawiki/2014/c/cc/Freiburg2014_Results_WB_receptorexpression_DNA_conc.jpg">
-
</figure>
+
      <figcaption>
-
</div>
+
        <p class="header">
-
</div>
+
          Figure 7: Tranfection of HEK-293T cells with different receptor DNA concentrations.
 +
        </p>
 +
        <p class="desc">
 +
          Cells (on 35mm plates) were transfected with 0.6 µg to 5.4 µg plasmid p14rz_006 coding
 +
          for mCAT-1-HA per well. Cells were lysed with RIPA buffer after 24 h of incubation and analyzed
 +
          by Western blotting. <a href="https://2014.igem.org/Team:Freiburg/Notebook/Labjournal#MirjaHarms_Expression_time_receptor">Labjournal</a>
 +
        </p>
 +
      </figcaption>
 +
    </figure>
 +
  </div>
 +
</div>
</section>
</section>
 +
<div class="row category-row">
 +
<div class="col-sm-6">
 +
<div class="container-fluid" style="float: left">
 +
<div style="position: relative; float: right; margin-top: 4px;">
 +
<a href="https://2014.igem.org/Team:Freiburg/Results/Vector">Go back to The Vector</div>
 +
<div style="position: relative; float: left;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/4/44/Freiburg2014_Navigation_Arrow_rv.png">  <!-- Pfeil rv--></a></div>
 +
</div>
 +
</div>
 +
<div class="col-sm-6">
 +
<div class="container-fluid" style="float: right">
 +
<div style="position: relative; float: left; margin-top: 4px;">
 +
<a href="https://2014.igem.org/Team:Freiburg/Results/Light_system">See the Results of our Ligth Systems</div>
 +
<div style="position: relative; float: right;"> <img class="img-no-border" style="max-width: 50px; margin-top:5px;" src=" https://static.igem.org/mediawiki/2014/9/95/Freibur2014_pfeilrechts.png">  <!-- Pfeil fw--></a></div>
 +
</div>
 +
</div>
 +
</div>

Latest revision as of 03:19, 18 October 2014

The AcCELLerator

The Receptor

The specificity of our system is based on the murine CAT-1 receptor, which serves as the viral entry site of our viral vector into the target cells. We demonstrate that the virus can be used to stably integrate genes into the genome of murine cells. We also targeted other cell lines by expressing the gene for the murine CAT-1 receptor in these cells before viral transduction. Since mCAT-1 is naturally only present in murine cell lines, we use human cell lines for transfection of the receptor. Expression of a reporter suggests that only the cells expressing the mCAT-1 receptor are being infected by the viral particles, thus making particular gene transfer possible.

Subcellular Localization of the 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. In order to determine the localization of the mCAT-1 receptor, we labeled the C-terminus of the mCAT-1 with the fluorescent protein mCherry and transfected it into human embryonic kidney (HEK-293T) cells. After distinct time points cells were imaged with a confocal scanning laser microscope. The mCAT1-mCherry construct was found predominantly at the surface of the cells (Figs. 1, 2).

Figure 1: Overview image of HEK-293T cells transfected with mCAT-1-mCherry (p14rz_005).

Confocal pictures were taken with a 20x plan apo objective. Nuclear staining (DAPI) is shown in blue and the mCAT-1-mCherry in red.Labjournal

Figure 2: HEK-293T cells transfected with mCAT-1-mCherry (p14rz_005).

Confocal pictures were taken with an 100x oil immersion objective (NA 1.40). Nuclear staining (DAPI) is shown in blue and the mCAT-1-mCherry in red.

Receptor Functionality and Virus Specificity

Transduction of genes into murine cell lines that naturally express the mCAT-1 receptor occurred with an efficiency of over 80%. For our system we needed functional expression of the receptor in non-murine cell lines, i.e. the receptor has to serve as an entry site for the virus, and the virus has to efficiently deliver its cargo into the target cell. We tested the functionality of the mCAT-1 receptor by expressing it in HEK-293T cells and subsequent infection with the virus containing EGFP as a cargo. The presence of green fluorescent cells in the infected cultures of different non-murine cell lines indicated that the receptor was not only expressed but can also be used as an entry site by the virus.

During our project we generated receptor constructs which were labeled with fluorescent protein (for microscopy) or the HA tag (for Western blotting). Since we didn't know whether the tags have an impact on viral infection capabilities, we tested them for their functionality, confirming that all receptor constructs lead to viral infection when expressed in non-murine cell lines (Fig. 3). For making the receptor visible for fluorescent microscopy and analysis by flow cytometry, we used the mCherry tag (constructs p14rz_005 and p14rz_006).

Figure 3: HEK-293T cells transfected with different receptor constructs and infected with MuLV-EGFP afterwards.

Cells transfected with (A) mCAT-1, (B) mCAT-1-HA and (C) mCAT-1-HA-mCherry (red). Cells were transduced with MuLV-EGFP after 24 h. Pictures were taken after 48 h. EGFP fluorescence is shown in green. Labjournal

Owing to the limited resolution and sensitivity of the cell culture microscope used for the images in Fig. 3, it was not clear whether the cells expressing the receptor were the same cells that were transduced by the virus and expressed EGFP (Fig. 3C). Therefore we obtained higher qualtiy images with a confocal microscope (Fig. 4). The cells were transfected with the mCAT-1-mCherry construct, 24h later infected by the virus containing EGFP, and imaged 48h later. We found many cells that expressed EGFP in the cytoplasm and the mCAT-1 receptor on the plasma membrane, whereas we did not find cells that expressed EGFP but not the receptor, suggesting that mCAT-1 expression is required for infection of the cells by the MuLV.

We also noticed that mCAT-1-mCherry was less present after transduction with the viral particles. Possible explanations are that the receptor is internalized after infection by a mechanism initiated by the virus, or that the expression rate of the receptor decreases after infection. Since the particles can only transfer their cargo into dividing cells, we often found pairs of cells that were infected by the viral vector or observed high expression of the receptor in cells next to targeted cells.

We were wondering whether receptor expression is indeed required for virus entry into the cell. Although we observed by confocal microscopy that many cells that expressed EGFP also expressed the receptor, we set out to correlate the expression levels of receptor and EGFP, i.e. the cargo of the virus. Therefore we used FACS analysis to quantify the levels of mCherry and EGFP fluorescence in all cells (Fig. 5). Although many cells that expressed the receptor were not transduced with EGFP, we found that all cells that expressed EGFP also expressed the receptor (Fig. 5, top right quadrant). Only a negligible number of cells showed EGFP expression but low mCherry fluorescence (Fig. 5, top left quadrant). This requirement for the presence of the receptor to allow efficient virus entry indicated that the MuLV is indeed highly specific for the mCAT-1 receptor.

Figure 4: HEK-293T cells expressing mCAT-1-mCherry transduced with MuLV-GFP.

(A) Overlay of all three channels, (B) DAPI, (C) EGFP and (D) mCAT-1-mCherry. Objective plan apo 60x, NA 1.40. Labjournal

Figure 5: FACS data of HEK-293T cells tranfected with the receptor and infected with MuLV-EGFP.

HEK-293T cells were transfected with the mCAT-1-mCherry and infected with MuLV-EGFP. Cells were analyzed by flow cytometry 48 h after infection. Labjournal

Receptor Expression

To enable efficient infection of cells with the virus, the time point for viral infection should match the time the receptor needs for expression in target cells. In order to determine the expression time of mCAT-1 in HEK-293T cells, we transfected the cells with the HA-tagged mCAT-1. Cells transfected with receptor DNA were analyzed after different incubation times. We found that the expression of the receptor peaked at 24 h after transfection (Fig. 6). In later experiments we used this time point for viral infections.

Since the presence of mCAT-1 on the cell surface is a main factor for viral tranduction efficiency, we quantified the expression of the receptor after transfection of HEK-293T cells with different concentrations of receptor DNA by Western blotting. We found that the expression of the receptor in the cell increases with higher amounts of transfected DNA (Fig. 7). However, we did not use such high DNA concentrations for our later experiments because we found that non-murine cells transfected with high amounts of transfected DNA died, probably due to overexpression of the mCAT-1 receptor.

Figure 6: Expression time of the receptor that was transfected into HEK-293T cells.

After transfection with mCAT-1-HA cells were lysed with RIPA buffer at distinct time points. A Western blot was performed using an anti-HA antibody. Labjournal

Figure 7: Tranfection of HEK-293T cells with different receptor DNA concentrations.

Cells (on 35mm plates) were transfected with 0.6 µg to 5.4 µg plasmid p14rz_006 coding for mCAT-1-HA per well. Cells were lysed with RIPA buffer after 24 h of incubation and analyzed by Western blotting. Labjournal