Difference between revisions of "Part:BBa K1470006"
m
 
(13 intermediate revisions by the same user not shown)
Line 5: Line 5:
 
<body>
 
<body>
  
−
<p> pMIG is a novel and easy way to handle mammalian expression vectors and to create fast and efficiently stable cell lines. It contains Long Terminal Repeats (LTR) for the production of viral vectors. There is no packaging signal or <i>gag, pol, env</i> in pMIG. Thus it can be used in any biosafety level 1 laboratory.</p>
+
<p> pMIG is a retroviral transfer plasmid to produce viral vectors when it is transfected into a packaging cell line. With these viral vectors it is possible to efficiently create stable cell lines in just one week. pMIG contains two long terminal repeats (5'LTR and 3'LTR), which flank a psi packaging sequence and space for genes with a size of up to 8 kb. The 5'LTR serves as a strong promoter for the gene of interest (GOI), that can be inserted with the restriction sites <i>Eco</i>RI and <i>Pst</i>I. This allows to use Biobricks of the RFC10 and RFC25 standard.
 +
 
 +
The originally existing genes for the envelope, the capsid proteins and the polymerase and integrase are located in a packaging cell line (Phoenix eco). By transfecting pMIG to this cell line, functional viral vectors containing the gene of interest are produced. The viral particles are secreted into the cell culture supernatant and can be used to infect target cells. The viral particles are derived from a Murine Leukemia Virus and therefore can only insert the gene of interest into the genome of dividing cells. <a href="http://2014.igem.org/Team:Freiburg/Project/The_viral_vector">Read more about the viral vector</a>
 +
 
  
 
<h4>Origin, Standardization and Application</h4>
 
<h4>Origin, Standardization and Application</h4>
 
<p>This plasmid was originally created by William Hahn and is available as plasmid 9044 at Addgene, we got his permission to submit pMIG as biobrick. It is a high copy plasmid and contains an Ampicillin resistance.</p>  
 
<p>This plasmid was originally created by William Hahn and is available as plasmid 9044 at Addgene, we got his permission to submit pMIG as biobrick. It is a high copy plasmid and contains an Ampicillin resistance.</p>  
  
−
<p>In order to provide this plasmid as a mammalian expression vector, we removed several EcoRI- and PstI-sites and an IRES. Afterwards we inserted a multiple cloning site, consisting of XhoI, EcoRI, NotI, SalI, PstI, HindHIII and ClaI. However, it was not possible to delete Xba I-sites in 3'- and 5' LTRs due to their nearly identical sequences. Nonetheless, any biobrick cut with EcoRI/PstI can be ligated in pMIG, hence providing future iGEM Teams a well functioning and reliable expression vector for mammalian cells.</p>
+
<p>In order to provide this plasmid as a mammalian expression vector, we removed several <i>Eco</i>RI- and <i>Pst</i>I-sites and an IRES. Afterwards we inserted a multiple cloning site, consisting of <i>Xho</i>I, <i>Eco</i>RI, <i>Not</i>I, <i>Sal</i>I, <i>Pst</i>I, <i>Hind</i>III and <i>Cla</i>I. However, it was not possible to delete <i>Xba</i>I sites in 3'LTR and 5'LTRs due to their nearly identical sequences. Nonetheless, any biobrick cut with <i>Eco</i>RI/<i>Pst</i>I can be ligated into pMIG, hence providing future iGEM Teams a well functioning and reliable expression vector for mammalian cells.</p>
 +
 
 +
<h4>Characterization</h4>
 +
<p>The viral vector uses the <a href="https://parts.igem.org/Part:BBa_K1470000">murine CAT-1 receptor (mCAT-1)</a> as an entry site into the cell. Therefore, it can specifically transduce murine cells (e.g. NIH3T3), but not cell lines from other species (e.g. the human embryonic kidney cells (HEK-293T).
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2014/7/79/Freiburg2014_Results_Specificity_MuLV_microscopy.jpg"  width="600px">
 +
<figcaption>
 +
<p><b>Figure 1: Infection of different cell lines with the viral vector derived from the murine leukemia virus.</b>
 +
Cells that were infected by viral particles express EGFP.</p>
 +
</figcaption>
 +
</figure>
 +
 
 +
<p>After transfection of human cells (HEK-293T) with the fusion protein mCAT-1-mCherry, viral vectors containing EGFP can also integrate into non-murine cells.</p>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2014/b/b0/Freiburg2014_Receptor_08_plus_Virus_single_channels.png"  width="600px">
 +
<figcaption>
 +
<p><b>Figure 2: HEK-293T cells expressing mCAT-1-mCherry transduced with MuLV-GFP.</b>
 +
(A) Overlay of all three channels, (B) DAPI, (C) EGFP and (D) mCAT-1-mCherry.</p>
 +
</figcaption>
 +
</figure>
 +
<br>
 +
<p>Different genes for fluorescent protein were inserted into pMIG. The produced viral vectors were used to infect murine NIH3T3 cells.</p>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/parts/f/f1/2014Freiburg_pMIG_transfection.png"  width="600px">
 +
<figcaption>
 +
<p><b>Figure 3: Murine NIH3T3 cells infected with our viral vector.</b>
 +
The vector contained different fluorescent proteins: (left) EGFP, (middle) mKO, (right) mKate that were stably integrated into the genome</p>
 +
</figcaption>
 +
</figure>
 +
<p>The viral vector can stably integrate genes. After the transduction we sorted positiv cells and passaged them 10 times. The amount of EGFP expressing cells were measured by FACS analysis.</p>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/parts/6/62/2014Freiburg_stable_cell_line.png"  width="600px">
 +
<figcaption>
 +
<p><b>Figure 4: Stable integration of EGFP into the genome of the target cells.</b>
 +
(left) Histogram of fluorescence intensity at the first sorting step. (middle) Histogram of fluorescence intensity after sorting. (right) The fraction of fluorescent cells stays constant for 10 passages.
 +
</p>
 +
</figcaption>
 +
</figure>
 +
 
 +
<a href="<a href="http://2014.igem.org/Team:Freiburg/Results/Vector">Read more about the results</a>
 +
<br>
 +
<br>
  
−
<p>Because of pMIG's retroviral origins, it can be used only in packaging cell lines such as Phoenix cells to produce viral vectors. Otherwise there will not be any functional vectors due to the lack of <i>gag, pol</i> and <i>env</i>.No additional promotor is required for expression on  the account of 3'LTR acting as one.</p>
+
<p>The workflow consists of the following steps: Transfection of the packaging cell line with pMIG containing the GOI, harvest of the viral vector in the cell culture supernatant and transduction of the target cells.</p>
−
<p>The viral vector uses the <a href="https://parts.igem.org/Part:BBa_K1470000">murine CAT-1 receptor (mCAT-1)</a> as an entry site into the cell. Therefore, it can specifically transduce mouse cells (e.g. NIH3T3), but not cells lines from other species (e.g. the human embryonic kidney (HEK-293T) cells). <a href="http://2014.igem.org/Team:Freiburg/Project/The_viral_vector"> More information</a>
+
 
<figure>
 
<figure>
−
<img scr="https://static.igem.org/mediawiki/2014/7/79/Freiburg2014_Results_Specificity_MuLV_microscopy.jpg">
+
<img src="https://static.igem.org/mediawiki/parts/8/87/2014Freiburg_workflow.png"  width="600px">
 
<figcaption>
 
<figcaption>
−
<b>Figure 1: Infection of different cell lines with the viral vector derived from the murine leukemia virus.</b>
+
<p><b>Figure 5: Workflow</b>.  
−
Cells that were infected by viral particles express EGFP.
+
</p>
 
</figcaption>
 
</figcaption>
 
</figure>
 
</figure>

Latest revision as of 02:26, 3 November 2014

pMIG

pMIG is a retroviral transfer plasmid to produce viral vectors when it is transfected into a packaging cell line. With these viral vectors it is possible to efficiently create stable cell lines in just one week. pMIG contains two long terminal repeats (5'LTR and 3'LTR), which flank a psi packaging sequence and space for genes with a size of up to 8 kb. The 5'LTR serves as a strong promoter for the gene of interest (GOI), that can be inserted with the restriction sites EcoRI and PstI. This allows to use Biobricks of the RFC10 and RFC25 standard. The originally existing genes for the envelope, the capsid proteins and the polymerase and integrase are located in a packaging cell line (Phoenix eco). By transfecting pMIG to this cell line, functional viral vectors containing the gene of interest are produced. The viral particles are secreted into the cell culture supernatant and can be used to infect target cells. The viral particles are derived from a Murine Leukemia Virus and therefore can only insert the gene of interest into the genome of dividing cells. Read more about the viral vector

Origin, Standardization and Application

This plasmid was originally created by William Hahn and is available as plasmid 9044 at Addgene, we got his permission to submit pMIG as biobrick. It is a high copy plasmid and contains an Ampicillin resistance.

In order to provide this plasmid as a mammalian expression vector, we removed several EcoRI- and PstI-sites and an IRES. Afterwards we inserted a multiple cloning site, consisting of XhoI, EcoRI, NotI, SalI, PstI, HindIII and ClaI. However, it was not possible to delete XbaI sites in 3'LTR and 5'LTRs due to their nearly identical sequences. Nonetheless, any biobrick cut with EcoRI/PstI can be ligated into pMIG, hence providing future iGEM Teams a well functioning and reliable expression vector for mammalian cells.

Characterization

The viral vector uses the murine CAT-1 receptor (mCAT-1) as an entry site into the cell. Therefore, it can specifically transduce murine cells (e.g. NIH3T3), but not cell lines from other species (e.g. the human embryonic kidney cells (HEK-293T).

Figure 1: Infection of different cell lines with the viral vector derived from the murine leukemia virus. Cells that were infected by viral particles express EGFP.

After transfection of human cells (HEK-293T) with the fusion protein mCAT-1-mCherry, viral vectors containing EGFP can also integrate into non-murine cells.

Figure 2: 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.


Different genes for fluorescent protein were inserted into pMIG. The produced viral vectors were used to infect murine NIH3T3 cells.

Figure 3: Murine NIH3T3 cells infected with our viral vector. The vector contained different fluorescent proteins: (left) EGFP, (middle) mKO, (right) mKate that were stably integrated into the genome

The viral vector can stably integrate genes. After the transduction we sorted positiv cells and passaged them 10 times. The amount of EGFP expressing cells were measured by FACS analysis.

Figure 4: Stable integration of EGFP into the genome of the target cells. (left) Histogram of fluorescence intensity at the first sorting step. (middle) Histogram of fluorescence intensity after sorting. (right) The fraction of fluorescent cells stays constant for 10 passages.

Read more about the results

The workflow consists of the following steps: Transfection of the packaging cell line with pMIG containing the GOI, harvest of the viral vector in the cell culture supernatant and transduction of the target cells.

Figure 5: Workflow.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 4917
    Illegal XbaI site found at 289
    Illegal XbaI site found at 3711
    Illegal PstI site found at 10
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 4917
    Illegal NheI site found at 99
    Illegal NheI site found at 3521
    Illegal PstI site found at 10
    Illegal NotI site found at 4925
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 4917
    Illegal BglII site found at 4899
    Illegal XhoI site found at 4905
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 4917
    Illegal XbaI site found at 289
    Illegal XbaI site found at 3711
    Illegal PstI site found at 10
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 4917
    Illegal XbaI site found at 289
    Illegal XbaI site found at 3711
    Illegal PstI site found at 10
    Illegal NgoMIV site found at 3466
    Illegal NgoMIV site found at 4123
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 520
    Illegal BsaI site found at 541
    Illegal BsaI site found at 3964
    Illegal BsaI.rc site found at 1901
    Illegal BsaI.rc site found at 4030
    Illegal BsaI.rc site found at 4645
    Illegal SapI site found at 818