Difference between revisions of "Part:BBa K1993009"
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<h2>'''Functions:'''</h2> | <h2>'''Functions:'''</h2> | ||
− | 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). | + | 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> | + | '''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]) | ||
− | < | + | <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]) | ||
− | <li>eGFP dramatically improved the spectral characteristics of GFP and allowed the practical use of | + | <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]) |
+ | |||
</ul> | </ul> | ||
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</table> | </table> | ||
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− | As for expression of eGFP, transfected | + | 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> | ||
<html><br> | <html><br> | ||
− | + | Figure 9:Under IVIS Spectrum, MSCs<sup>CXCR4</sup> could be detected and located in IBD mice.<br> | |
− | Under IVIS Spectrum, | + | |
<br> | <br> | ||
<li><br> | <li><br> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>Figure | + | <td>Figure 14</td> |
− | <td>Figure | + | <td>Figure 15</td> |
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− | <td>Figure | + | <td>Figure 16</td> |
− | <td>Figure | + | <td>Figure 17</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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− | 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. | + | 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
-
Figure 2 Figure 3 -
Figure 4 Figure 5 -
Figure 6 Figure 7
In vivo
Figure8 :Under IVIS Spectrum, MSCsCXCR4 exhibited enhanced capacities for targeted migration to the bowels in IBD model.
Figure 9:Under IVIS Spectrum, MSCsCXCR4 could be detected and located in IBD mice.
Figure 10 Figure 11 Figure 12 Figure 13
Figure 14 Figure 15 Figure 16 Figure 17
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 FeaturesAssembly Compatibility:- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1350
Illegal BamHI site found at 494
Illegal BamHI site found at 1893 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
- 10