Difference between revisions of "Part:BBa K1993009"
Line 3: | Line 3: | ||
<partinfo>BBa_K1993009 short</partinfo> | <partinfo>BBa_K1993009 short</partinfo> | ||
− | + | <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). | ||
+ | <html> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/b/b1/T--SYSU-MEDICINE--1.2.15.png" style="width:400px" ></a> | ||
+ | |||
+ | </html> | ||
+ | |||
+ | '''Figure 1 CXCR4-T2A-Luciferase-IRES-eGFP'''<br> | ||
+ | |||
+ | <h2>'''Details:'''</h2> | ||
+ | <ul> | ||
+ | <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]) | ||
+ | <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]) | ||
+ | <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>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]) | ||
+ | </ul> | ||
+ | |||
+ | <h2>'''Results:'''</h2> | ||
+ | |||
+ | <h3>In vitro </h3> | ||
+ | <ol> | ||
+ | <li> | ||
+ | <html> | ||
+ | <table > | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2016/a/a8/T--SYSU-MEDICINE--2.2.2.png" style="width:200px" ></a></td> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2016/7/78/T--SYSU-MEDICINE--wbCXCR4.jpg" style="width:200px" ></a></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Figure 2</td> | ||
+ | <td>Figure 3</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </html> | ||
+ | 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. | ||
+ | <li> | ||
+ | <html> | ||
+ | <table > | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2016/9/94/T--SYSU-MEDICINE--eGFP-293T-CXCR4.jpg" style="width:200px;height:200px" ></a></td> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2016/f/fd/T--SYSU-MEDICINE--eGFP-MSC-CXCR4.jpg" style="width:200px;height:200px" ></a></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Figure 4</td> | ||
+ | <td>Figure 5</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </html> | ||
+ | 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. | ||
+ | |||
+ | <li> | ||
+ | <html> | ||
+ | <table > | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2016/4/4e/T--SYSU-MEDICINE--2.3.2.png" style="width:300px" ></a></td> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2016/2/2f/T--SYSU-MEDICINE--cell-CXCR4-eGFP.jpg" style="width:100px" ></a></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Figure 6</td> | ||
+ | <td>Figure 7</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </html> | ||
+ | Chemotaxis of engineered MSCs were examined against CXCL12. Results were reported as number of cells migrated. Chemotaxis capacity of our engineered MSCs improved. | ||
+ | </ol> | ||
+ | <h3>In vivo</h3> | ||
+ | <ol> | ||
+ | <li><br> | ||
+ | <html> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/a/a8/T--SYSU-MEDICINE--2.5.4.1.jpg" style="height:200px"></a> | ||
+ | <html><br> | ||
+ | Figure8 :Under IVIS Spectrum, MSCsCXCR4 exhibited enhanced capacities for targeted migration to the bowels in IBD model. | ||
+ | |||
+ | <li> | ||
+ | |||
+ | "'Figure 9"' | ||
+ | Under IVIS Spectrum, MSCsCXCR4 could be detected and located in IBD mice. | ||
+ | |||
+ | 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. In a word, MSCsCXCR4 have greater immunoregulatory function. | ||
+ | |||
+ | |||
+ | |||
+ | 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. In a word, MSCsCXCR4 have greater immunoregulatory function. | ||
+ | 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. | ||
+ | <br> | ||
+ | <br> | ||
+ | <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 10:50, 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 (IBD).
Figure 1 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) <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 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 GFPs in mammalian cells. (Details could be seen on [htttp://parts.igem.org/Part:BBa_K1993017 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.
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. In a word, MSCsCXCR4 have greater immunoregulatory function.
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. In a word, MSCsCXCR4 have greater immunoregulatory function.
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 FeaturesBBa_K1993009 SequenceAndFeatures