Difference between revisions of "Part:BBa K1993028"

 
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<partinfo>BBa_K1993028 short</partinfo>
 
<partinfo>BBa_K1993028 short</partinfo>
  
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CXCR5-IRES-eGFP
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<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_K1993008 under the control of EF-1α. (Figure 1) After that, plasmid BBa_K1993008 would be transduced into MSCs by lentivirus expression vector. What’s more, we confirmed its function in vitro and in vivo (delayed type hypersensitivity animal models, DTH).
  
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<html>
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<img src="https://static.igem.org/mediawiki/2016/b/b1/T--SYSU-MEDICINE--1.2.16.png" style="width:400px"  ></a>
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</html>
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'''Figure 1  EF-1α-CXCR5-IRES-eGFP'''<br>
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<h2>'''Details:'''</h2>
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<ul>
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<li>Elongation factor-1α (EF-1α), a constitutive promoter, is responsible for mediating the recruitment of aminoacyl-tRNA to the A-site of the 80S ribosome during protein synthesis.
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<li>CXCR5 is a chemokine receptor and its corresponding ligand is CXCL13. (Details could be seen on [https://parts.igem.org/Part:BBa_K1993013 BBa_K1993013])
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<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 GFP in mammalian cells. (Details could be seen on [https://parts.igem.org/Part:BBa_K1993017 BBa_K1993017])
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</ul>
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<h2>'''Results:'''</h2>
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<h3>In vitro </h3>
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<ol>
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<li>
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<html>
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<table >
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  <tr>
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    <td><img src="https://static.igem.org/mediawiki/2016/7/74/T--SYSU-MEDICINE--2.2.3.png" style="width:200px"  ></a></td>
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    <td><img src="https://static.igem.org/mediawiki/2016/f/ff/T--SYSU-MEDICINE--wbCXCR5.jpg" style="width:200px" ></a></td>
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  </tr>
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  <tr>
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    <td>Figure 2</td>
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    <td>Figure 3</td>
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  </tr>
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</table>
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</html>
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mRNA and protein levels of CXCR5 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 CXCR5 both elevated in our modified MSCs.
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<li>
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<html>
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<table >
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  <tr>
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    <td><img src="https://static.igem.org/mediawiki/2016/f/ff/T--SYSU-MEDICINE--293T-CXCR5-eGFP.png" style="width:200px;height:200px" ></a></td>
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    <td><img src="https://static.igem.org/mediawiki/2016/7/78/T--SYSU-MEDICINE--MSC-CXCR5-eGFP.png" style="width:200px;height:200px" ></a></td>
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  </tr>
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  <tr>
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    <td>Figure 4</td>
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    <td>Figure 5</td>
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  </tr>
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</table>
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</html>
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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 were expressed.
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<li>
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<html>
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<table >
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  <tr>
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    <td><img src="https://static.igem.org/mediawiki/2016/7/78/T--SYSU-MEDICINE--BBa_K1993008-fig6.png" style="width:300px"  ></a></td>
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    <td><img src="https://static.igem.org/mediawiki/2016/c/c4/T--SYSU-MEDICINE--BBa_K1993008-fig7.png" style="width:100px" ></a></td>
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  </tr>
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  <tr>
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    <td>Figure 6</td>
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    <td>Figure 7</td>
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  </tr>
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</table>
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</html>
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Chemotaxis of engineered MSCs were examined against CXCL13. Results were reported as number of cells migrated. Chemotaxis capacity of our engineered MSCs had improved.
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</ol>
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<h3>In vivo</h3>
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<ol>
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<li><br>
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<html>
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<img src="https://static.igem.org/mediawiki/2016/7/7e/T--SYSU-MEDICINE--BBa_K1993008-fig8.png" style="height:400px"></a>
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</html>
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<br>
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'''Figure8'''
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<br>
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Figure8 :The inflamed ears were collected from each group and subjected to in situ immunofluorescence staining. Our results revealed that MSC<sup>CXCR5</sup> exhibited enhanced capacities for targeted migration to the ears in DTH model.
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<li><br>
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<html>
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<img src="https://static.igem.org/mediawiki/2016/7/74/T--SYSU-MEDICINE--BBa_K1993008-fig9.png" style="heigh:t100px"></a>
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</html>
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<br>
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'''Figure9'''
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<br>
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The inflamed ears were collected from each group, subjected to in situ immunofluorescence staining and observed under fluorescence microscope. Our results revealed MSC<sup>CXCR5</sup> could be located in the inflamed ears of DTH mice.
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<br>
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<li><br>
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<html>
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<img src="https://static.igem.org/mediawiki/2016/0/0d/T--SYSU-MEDICINE--BBa_K1993008-fig10.png"  style="width:600px"  ></a>
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</html>
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<br>
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'''Figure 10'''
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<br>
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Ear sampling for quantitative PCR analysis. Comparing with DTH+MSCs group, levels of anti-inflammatory cytokines (IL-10) elevated in DTH+MSC<sup>CXCR5</sup> group, while levels of pro-inflammatory cytokines (IL-4, TNF-α, IFN-γ, IL-6 and IL-17) decreased in DTH+MSC<sup>CXCR5</sup> group.
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'''In a word, MSC<sup>CXCR5</sup> displayed greater immunoregulatory effect.'''
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<li><br>
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<html>
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<img src="https://static.igem.org/mediawiki/2016/7/79/T--SYSU-MEDICINE--BBa_K1993008-fig11.png"  style="width:500px"  ></a>
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</html>
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<br>
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'''Figure 11'''
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<br>
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<html>
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<img src="https://static.igem.org/mediawiki/2016/c/c6/T--SYSU-MEDICINE--BBa_K1993008-fig12.png"  style="width:500px"  ></a>
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</html>
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<br>
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'''Figure 12'''
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<br>
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After injection, MSC<sup>CXCR5</sup> displayed better treatment efficacy than MSC<sup>eGFP</sup>, characterized by their abilities to decrease ear thickness and leukocyte infiltration. MSC<sup>CXCR5</sup> significantly attenuated DTH as early as 24 hours post-injection, and had even greater effects at 48 hours post-injection.
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'''In a word, MSC<sup>CXCR5</sup> regulated immune system in inflammatory condition in a more significant way.'''
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<br>
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<br>
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<br>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 09:43, 18 October 2016


CXCR5-IRES-eGFP

Functions:

With the purpose of increasing homing efficiency of MSCs and illustrating their distribution, we constructed a plasmid BBa_K1993008 under the control of EF-1α. (Figure 1) After that, plasmid BBa_K1993008 would be transduced into MSCs by lentivirus expression vector. What’s more, we confirmed its function in vitro and in vivo (delayed type hypersensitivity animal models, DTH).

Figure 1 EF-1α-CXCR5-IRES-eGFP

Details:

  • Elongation factor-1α (EF-1α), a constitutive promoter, is responsible for mediating the recruitment of aminoacyl-tRNA to the A-site of the 80S ribosome during protein synthesis.
  • CXCR5 is a chemokine receptor and its corresponding ligand is CXCL13. (Details could be seen on BBa_K1993013)
  • 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 CXCR5 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 CXCR5 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 were expressed.
  3. Figure 6 Figure 7
    Chemotaxis of engineered MSCs were examined against CXCL13. Results were reported as number of cells migrated. Chemotaxis capacity of our engineered MSCs had improved.

In vivo



  1. Figure8
    Figure8 :The inflamed ears were collected from each group and subjected to in situ immunofluorescence staining. Our results revealed that MSCCXCR5 exhibited enhanced capacities for targeted migration to the ears in DTH model.


  2. Figure9
    The inflamed ears were collected from each group, subjected to in situ immunofluorescence staining and observed under fluorescence microscope. Our results revealed MSCCXCR5 could be located in the inflamed ears of DTH mice.


  3. Figure 10
    Ear sampling for quantitative PCR analysis. Comparing with DTH+MSCs group, levels of anti-inflammatory cytokines (IL-10) elevated in DTH+MSCCXCR5 group, while levels of pro-inflammatory cytokines (IL-4, TNF-α, IFN-γ, IL-6 and IL-17) decreased in DTH+MSCCXCR5 group. In a word, MSCCXCR5 displayed greater immunoregulatory effect.


  4. Figure 11

    Figure 12
    After injection, MSCCXCR5 displayed better treatment efficacy than MSCeGFP, characterized by their abilities to decrease ear thickness and leukocyte infiltration. MSCCXCR5 significantly attenuated DTH as early as 24 hours post-injection, and had even greater effects at 48 hours post-injection. In a word, MSCCXCR5 regulated immune system in inflammatory condition in a more significant way.


    Sequence and Features

    Assembly Compatibility:
    • 10
      COMPATIBLE WITH RFC[10]
    • 12
      COMPATIBLE WITH RFC[12]
    • 21
      INCOMPATIBLE WITH RFC[21]
      Illegal XhoI site found at 31
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      INCOMPATIBLE WITH RFC[25]
      Illegal NgoMIV site found at 973
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal BsaI.rc site found at 259
      Illegal SapI site found at 1050