Difference between revisions of "Part:BBa K1993009"
 
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<h2>'''Functions:'''</h2>
 
<h2>'''Functions:'''</h2>
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With the purpose of increasing homing efficiency of MSCs and illustrating their distribution, we constructed a plasmid BBa_K1993009 under the control of EF-1α. (Figure 1) After that, plasmid BBa_K1993009 would be transduced into MSCs by lentivirus expression vector. What’s more, we would confirm its function in vitro and in vivo (IBD).   
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With the purpose of increasing homing efficiency of MSCs and illustrating their distribution, we constructed a plasmid BBa_K1993009 under the control of EF-1α. (Figure 1) After that, plasmid BBa_K1993009 would be transduced into MSCs by lentivirus expression vector. What’s more, we would confirm its function in vitro and in vivo (inflammatory bowel disease animal models, IBD).   
  
 
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'''Figure 1  CXCR4-T2A-Luciferase-IRES-eGFP'''<br>
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'''Figure 1  EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP'''<br>
  
 
<h2>'''Details:'''</h2>
 
<h2>'''Details:'''</h2>
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<li>Elongation factor-1α (EF-1α), as a constitutive promoter, is responsible for mediating the recruitment of aminoacyl-tRNA to the A-site of the 80S ribosome during protein synthesis.
 
<li>Elongation factor-1α (EF-1α), as a constitutive promoter, is responsible for mediating the recruitment of aminoacyl-tRNA to the A-site of the 80S ribosome during protein synthesis.
 
<li> CXCR4 is a chemokine receptor and its corresponding ligand is CXCL12. (Details could be seen on [https://parts.igem.org/Part:BBa_K1993003 BBa_K1993003])  
 
<li> CXCR4 is a chemokine receptor and its corresponding ligand is CXCL12. (Details could be seen on [https://parts.igem.org/Part:BBa_K1993003 BBa_K1993003])  
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<lT2A is a self-cleaving 2A peptide (The average length is 18–22 amino acids) that could be applied in expression of more than one gene in cells. (Details could be seen on [https://parts.igem.org/Part:BBa_K1993019 BBa_K1993019])
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<li>T2A is a self-cleaving 2A peptide (The average length is 18–22 amino acids) that could be applied in expression of more than one gene in cells. (Details could be seen on [https://parts.igem.org/Part:BBa_K1993019 BBa_K1993019])
 
<li>Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is distinct from a photoprotein. (Details could be seen on [https://parts.igem.org/Part:BBa_K1993015 BBa_K1993015])
 
<li>Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is distinct from a photoprotein. (Details could be seen on [https://parts.igem.org/Part:BBa_K1993015 BBa_K1993015])
 
<li>Internal ribosome entry site (IRES) is an RNA element that allows for translation initiation in an end-independent manner, as part of the greater process of protein synthesis. (Details could be seen on [https://parts.igem.org/Part:BBa_K1993016 BBa_K1993016])
 
<li>Internal ribosome entry site (IRES) is an RNA element that allows for translation initiation in an end-independent manner, as part of the greater process of protein synthesis. (Details could be seen on [https://parts.igem.org/Part:BBa_K1993016 BBa_K1993016])
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<li>eGFP dramatically improved the spectral characteristics of GFP and allowed the practical use of GFPs in mammalian cells. (Details could be seen on [htttp://parts.igem.org/Part:BBa_K1993017 BBa_K1993017])
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<li>eGFP dramatically improved the spectral characteristics of GFP and allowed the practical use of GFP in mammalian cells. (Details could be seen on [https://parts.igem.org/Part:BBa_K1993017 BBa_K1993017])
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As for expression of eGFP, transfected 293T cells (Figure 4) and MSCs (Figure 5) were observed under fluorescent microscope. As a result, eGFP were expressed.
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As for expression of eGFP, transfected 293FT cells (Figure 4) and MSCs (Figure 5) were observed under fluorescent microscope. As a result, eGFP was expressed.
  
 
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<img src="https://static.igem.org/mediawiki/2016/e/e5/T--SYSU-MEDICINE--3.2.6和3.3.6.jpg" style="height:200px"></a>
 
<img src="https://static.igem.org/mediawiki/2016/e/e5/T--SYSU-MEDICINE--3.2.6和3.3.6.jpg" style="height:200px"></a>
 
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"'Figure 9"'<br>
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Figure 9:Under IVIS Spectrum, MSCs<sup>CXCR4</sup> could be detected and located in IBD mice.<br>
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Under IVIS Spectrum, MSCsCXCR4 could be detected and located in IBD mice.
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Colon sampling for quantitative PCR analysis. Comparing with TNBS+MSC group, levels of the anti-inflammatory cytokine (IL-10) elevated in TNBS+MSC<sup>CXCR4</sup> group while pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) levels decreased in TNBS+ MSC<sup>CXCR4</sup> group. In a word, MSCsCXCR4 have greater immunoregulatory function.
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Colon sampling for quantitative PCR analysis. Comparing with TNBS+MSC group, levels of the anti-inflammatory cytokine (IL-10) elevated in TNBS+MSC<sup>CXCR4</sup> group while pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) levels decreased in TNBS+ MSC<sup>CXCR4</sup> group.  
  
 
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     <td>Figure 10</td>
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     <td>Figure 14</td>
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     <td>Figure 11</td>
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     <td>Figure 15</td>
 
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     <td>Figure 12</td>
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     <td>Figure 16</td>
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     <td>Figure 13</td>
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     <td>Figure 17</td>
 
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After injecting with MSCs<sup>CXCR4</sup>, survival rate, body weight, length of colon and DAI score of IBD mice were improved significantly comparing to TNBS+MSC group and TNBS+saline group. In a word, MSCs<sup>CXCR4</sup>have greater immunoregulatory function.
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After injecting with MSCs<sup>CXCR4</sup>, survival rate, body weight, length of colon and DAI score of IBD mice were improved significantly comparing to TNBS+MSC group and TNBS+saline group.  
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Photographs of hematoxylin/eosin-stained colon cross-sections 2 days after injecting MSCs. MSCsCXCR4 displayed better treatment efficacy than TNBS+MSC group and TNBS+saline group, characterized by their abilities to decrease leukocyte infiltration.  
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Figure 18 Photographs of hematoxylin/eosin-stained colon cross-sections 2 days after injecting MSCs. MSCs<sup>CXCR4</sup> displayed better treatment efficacy than TNBS+MSC group and TNBS+saline group, characterized by their abilities to decrease leukocyte infiltration.  
 
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Latest revision as of 18:28, 17 October 2016


CXCR4-T2A-Luciferase-IRES-eGFP

Functions:

With the purpose of increasing homing efficiency of MSCs and illustrating their distribution, we constructed a plasmid BBa_K1993009 under the control of EF-1α. (Figure 1) After that, plasmid BBa_K1993009 would be transduced into MSCs by lentivirus expression vector. What’s more, we would confirm its function in vitro and in vivo (inflammatory bowel disease animal models, IBD).

Figure 1 EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP

Details:

  • Elongation factor-1α (EF-1α), as a constitutive promoter, is responsible for mediating the recruitment of aminoacyl-tRNA to the A-site of the 80S ribosome during protein synthesis.
  • CXCR4 is a chemokine receptor and its corresponding ligand is CXCL12. (Details could be seen on BBa_K1993003)
  • T2A is a self-cleaving 2A peptide (The average length is 18–22 amino acids) that could be applied in expression of more than one gene in cells. (Details could be seen on BBa_K1993019)
  • Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is distinct from a photoprotein. (Details could be seen on BBa_K1993015)
  • Internal ribosome entry site (IRES) is an RNA element that allows for translation initiation in an end-independent manner, as part of the greater process of protein synthesis. (Details could be seen on BBa_K1993016)
  • eGFP dramatically improved the spectral characteristics of GFP and allowed the practical use of GFP in mammalian cells. (Details could be seen on BBa_K1993017)

Results:

In vitro

  1. Figure 2 Figure 3
    mRNA and protein levels of CXCR4 of MSCs and our modified MSCs were semi-quantified and detected by qPCR and western blot, respectively. mRNA (Figure 2) and protein levels (Figure 3) of CXCR4 both elevated in our modified MSCs.
  2. Figure 4 Figure 5
    As for expression of eGFP, transfected 293FT cells (Figure 4) and MSCs (Figure 5) were observed under fluorescent microscope. As a result, eGFP was expressed.
  3. Figure 6 Figure 7
    Chemotaxis of engineered MSCs were examined against CXCL12. Results were reported as number of cells migrated. Chemotaxis capacity of our engineered MSCs improved.

In vivo



  1. Figure8 :Under IVIS Spectrum, MSCsCXCR4 exhibited enhanced capacities for targeted migration to the bowels in IBD model.


  2. Figure 9:Under IVIS Spectrum, MSCsCXCR4 could be detected and located in IBD mice.


  3. Figure 10 Figure 11
    Figure 12 Figure 13
    Colon sampling for quantitative PCR analysis. Comparing with TNBS+MSC group, levels of the anti-inflammatory cytokine (IL-10) elevated in TNBS+MSCCXCR4 group while pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) levels decreased in TNBS+ MSCCXCR4 group.

  4. Figure 14 Figure 15
    Figure 16 Figure 17
    After injecting with MSCsCXCR4, survival rate, body weight, length of colon and DAI score of IBD mice were improved significantly comparing to TNBS+MSC group and TNBS+saline group.


  5. Figure 18 Photographs of hematoxylin/eosin-stained colon cross-sections 2 days after injecting MSCs. MSCsCXCR4 displayed better treatment efficacy than TNBS+MSC group and TNBS+saline group, characterized by their abilities to decrease leukocyte infiltration.

    In a word, MSCsCXCR4 have greater immunoregulatory function.


    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 1350
      Illegal BamHI site found at 494
      Illegal BamHI site found at 1893
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      COMPATIBLE WITH RFC[25]
    • 1000
      COMPATIBLE WITH RFC[1000]