Difference between revisions of "Part:BBa K1993005"
 
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<h2>'''Functions:'''</h2>
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<img src="https://static.igem.org/mediawiki/2016/2/22/T--SYSU-MEDICINE--BBa_K1071009-fig1.jpg" style="width:400px"  ></a>
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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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In consideration of testifying the location of MSCs in multiple ways and provide future teams with optional and economic way to observe, we enhanced the basic part of RLuc with the gene of eGFP and improved a previous part, which was constructed by  iGEM13_Marburg . (For more details, See [https://parts.igem.org/Part:BBa_K1071009 BBa_K1071009]) .
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eGFP is a type of GFP derivatives by mutation. This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability, and a shift of the major excitation peak to 488 nm, with the peak emission kept at 509 nm. eGFP is one of the most used fluorescent proteins which is easy to detect. In our experiments, we applied eGFP in order to report the expression of Luciferase and to show the actual sites of MSCs in vitro.
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'''Figure 1  EF-1α-CXCR5-IRES-eGFP'''<br>
  
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<h2>'''Details:'''</h2>
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Another marking protein we engineered to MSCs is Luciferase. Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence by oxidizing luciferin substrate and responsible for regulating light production in a variety of organs. It’s convenient to observe biological processes, especially allowing for observation of cells non-invasively and specifically. In this composite part, we chose the gene of Renilla-luciferin 2-monooxygenase (RLuc) (936 bp). Unlike Firefly Luciferase (Fluc)（Details can be seen from [https://parts.igem.org/Part:BBa_K1993018 BBa_K1993018]) requiring ATP to catalyze, RLuc is a shorter version cooperating with O2 to oxidize luciferin. When MSCs engineered with coding sequence of Luciferase were administrated to mice that were injected intraperitoneally with Luciferin simultaneously, those areas accumulated with higher concentration of MSCs would oxidize more luciferin therefore display stronger fluorescence in IVIS spectrum system.  
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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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  <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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    <td>Figure 2</td>
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    <td>Figure 3</td>
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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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    <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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    <td>Figure 4</td>
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    <td>Figure 5</td>
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  </tr>
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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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On one hand, the advantages of eGFP lie in convenience as well as low cost for observation comparing to Luciferase. On the other hand, Luciferase make up for the shortage of eGFP as oxidized Luciferin could be observed in living animals， which expands its application range and lays a foundation for its further clinical application. The observation of these two complementary marking proteins ensures accurate locating of MSCs both in vitro and in vivo.
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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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    <td>Figure 6</td>
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    <td>Figure 7</td>
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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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To ensure co-expression of Luciferase and eGFP, we introduced internal ribosome entry site (IRES) （Details can be seen from [https://parts.igem.org/Part:BBa_K1993016 BBa_K1993016]) between them, an RNA element that allows for translation initiation in an end-independent manner, as part of the greater process of protein synthesis. In eukaryotic translation, initiation typically occurs at the 5' end of mRNA molecules, since 5' cap recognition is required for the assembly of the initiation complex. The location for IRES elements is often in the 5'UTR, but can also occur elsewhere in mRNAs.
 
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IRES has been widely used due to the following advantages: (1) ensured co-expression of genes before and after the IRES; (2) feasibility of adding subcellular localization sequences to the gene after IRES; and (3) availability of commercial expression plasmids harboring IRES. [2]
 
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In our project, firstly we found that protein coding genes downstream were not expressed if they followed another protein coding gene directly. Then we found the fantastic function of IRES and purified it (Figure 1). Taking advantage of function of IRES, we constructed a plasmid that IRES linked between two protein coding genes. For example, we constructed [https://parts.igem.org/Part:BBa_K1993001 BBa_K1993005] (Luciferase-IRES-eGFP) to ensure eGFP could be expressed.
 
  
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In our experiments, we confirmed its expression by fluorescent microscope and IVIS spectrum system. The results are shown as follows:
 
  
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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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'''Figure8'''
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<img src="https://static.igem.org/mediawiki/2016/7/75/T--SYSU-MEDICINE--BBa_K1071009-fig3.png" style="width:300px"  ></a>
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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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<p align="center">'''Figure1'''</p><br>
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We first testified the expression of eGFP in 293FT cells. As a result, eGFP had been successfully transduced into 293FT cells. (Figure 1)
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<p align="center">'''Figure2.'''</p>
 
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We transfected MSCs with our composite part. Figure 2 indicates successful expression of eGFP in MSCs.
 
  
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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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'''Figure9'''
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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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<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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'''Figure 10'''
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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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<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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'''Figure 11'''
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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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'''Figure 12'''
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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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<img src="https://static.igem.org/mediawiki/2016/6/68/T--SYSU-MEDICINE--BBa_K1071009-fig4.png" style="width:300px"  ></a>
 
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<p align="center">'''Figure3.'''</p>
 
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After intraperitoneal injection of Luciferase, MSCs engineered with Luciferase and eGFP were applied to mouse. Figure 3 showed the signal of oxidized Luciferin in living mice.
 
  
  

Latest revision as of 16:46, 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