Difference between revisions of "Part:BBa K1470006"
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<body>
 
<body>
  
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<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>
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<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, 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.
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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.
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Because of pMIG's retroviral origin, it is 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>
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<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>  
  
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<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>
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<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 EcoRI/PstI can be ligated into pMIG, hence providing future iGEM Teams a well functioning and reliable expression vector for mammalian cells.</p>
  
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<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>
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<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). <a href="http://2014.igem.org/Team:Freiburg/Project/The_viral_vector"> More information</a>
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<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>
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<figure>
 
<figure>
 
<img src="https://static.igem.org/mediawiki/2014/7/79/Freiburg2014_Results_Specificity_MuLV_microscopy.jpg"  width="600px">
 
<img src="https://static.igem.org/mediawiki/2014/7/79/Freiburg2014_Results_Specificity_MuLV_microscopy.jpg"  width="600px">

Revision as of 00:55, 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, 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. Because of pMIG's retroviral origin, it is 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 gag, pol and env.No additional promotor is required for expression on the account of 3'LTR acting as one.

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.

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). More information

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.

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