Difference between revisions of "Part:BBa K3113101"

 
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mGag is short for mouse Gag. This sequence codes for the coat protein of the human immunodeficiency virus optimised to bud in mice. It mediates the essential events in virion assembly, including binding the plasma membrane, making the protein-protein interactions necessary to create spherical particles.
 
mGag is short for mouse Gag. This sequence codes for the coat protein of the human immunodeficiency virus optimised to bud in mice. It mediates the essential events in virion assembly, including binding the plasma membrane, making the protein-protein interactions necessary to create spherical particles.
  
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<h2>Usage and Biology</h2>
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===Usage and Biology===
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While exosome secretion is a ubiquitous mechanism, HIV Gag vesicle formation is a bioorthogonal mechanism in mice. This poses a problem because it is known that small animal models have several cellular barriers to prevent HIV replication. In mice, assembly and budding of HIV vesicles are blocked<ref>Sherer, N.M., Swanson, C.M., Papaioannou, S., and Malim, M.H. (2009). Matrix Mediates the Functional Link between Human Immunodeficiency Virus Type 1 RNA Nuclear Export Elements and the Assembly Competency of Gag in Murine Cells. J. Virol. 83, 8525–8535.
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</ref>, thus severely interfering with the usage of Gag vesicles for our purposes. However, publications report different modifications to overcome these limitations<ref>Quellen</ref>. We, therefore, codon-optimized our Gag construct for expression in mouse cells and introduced the point mutation L21S<ref>Diaz-Griffero, F., Taube, R., Muehlbauer, S.M., and Brojatsch, J. (2008). Efficient production of HIV-1 viral-like particles in mouse cells. Biochem. Biophys. Res. Commun. 368, 463–469.</ref> to aid vesicle formation and secretion. As can be seen in Figure X, we succeeded in secreting Gag vesicles from MIN6-K8 as shown by HiBiT measurements. Introducing the L21S point mutation almost doubled the absolute amount of secreted vesicles compared to the wildtype Gag construct, while relative secretion rates were robustly above 40%.
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<h2>Characterization</h2>
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<partinfo>BBa_K3113101 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3113101 SequenceAndFeatures</partinfo>
  
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<h2>References</h2>
  
 
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Revision as of 13:41, 21 October 2019


mGag

mGag is short for mouse Gag. This sequence codes for the coat protein of the human immunodeficiency virus optimised to bud in mice. It mediates the essential events in virion assembly, including binding the plasma membrane, making the protein-protein interactions necessary to create spherical particles.

Usage and Biology

While exosome secretion is a ubiquitous mechanism, HIV Gag vesicle formation is a bioorthogonal mechanism in mice. This poses a problem because it is known that small animal models have several cellular barriers to prevent HIV replication. In mice, assembly and budding of HIV vesicles are blocked[1], thus severely interfering with the usage of Gag vesicles for our purposes. However, publications report different modifications to overcome these limitations[2]. We, therefore, codon-optimized our Gag construct for expression in mouse cells and introduced the point mutation L21S[3] to aid vesicle formation and secretion. As can be seen in Figure X, we succeeded in secreting Gag vesicles from MIN6-K8 as shown by HiBiT measurements. Introducing the L21S point mutation almost doubled the absolute amount of secreted vesicles compared to the wildtype Gag construct, while relative secretion rates were robustly above 40%.

Characterization


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 779
    Illegal BglII site found at 1307
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 709
  • 1000
    COMPATIBLE WITH RFC[1000]

References

  1. ↑ Sherer, N.M., Swanson, C.M., Papaioannou, S., and Malim, M.H. (2009). Matrix Mediates the Functional Link between Human Immunodeficiency Virus Type 1 RNA Nuclear Export Elements and the Assembly Competency of Gag in Murine Cells. J. Virol. 83, 8525–8535.
  2. ↑ Quellen
  3. ↑ Diaz-Griffero, F., Taube, R., Muehlbauer, S.M., and Brojatsch, J. (2008). Efficient production of HIV-1 viral-like particles in mouse cells. Biochem. Biophys. Res. Commun. 368, 463–469.