Difference between revisions of "Part:BBa K1993001"
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<partinfo>BBa_K1993001 short</partinfo>
 
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Chemokine receptors are receptors found on the surface of certain cells that interact with chemokines. They have a 7-transmembrane (7-TM) structure and couple to G-protein for signal transduction within a cell. [1] (Figure 1) Following interaction with their specific chemokine ligands, chemokine receptors trigger a flux intracellular calcium (Ca2+) ions, initiate chemotaxis and guide the cell to a desired location. (Figure 2)
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Chemokine receptors are found on the surface of certain cells that interact with chemokines. They have a 7-transmembrane (7TM) structure and couples to G-protein for signal transduction within a cell[1] (Figure 1). Following interaction with their specific chemokine ligands, chemokine receptors trigger a flux inintracellular calcium (Ca2+) ions which initiate the onset of chemotaxis that traffics the cell to a desired location (Figure 2).
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'''Figure 1. typical structure of a chemokine receptor.'''
 
'''Figure 1. typical structure of a chemokine receptor.'''
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<img src="https://static.igem.org/mediawiki/2016/0/02/T--SYSU-MEDICINE--interaction.png" style="width:800px"  ></a>
  
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==References==
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[1]Allen, Samantha J.; Crown, Susan E.; Handel, Tracy M. (2007-01-01). "Chemokine: receptor structure, interactions, and antagonism". Annual Review of Immunology. 25: 787–820.
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[2] Griffith J W, Sokol C L, Luster A D. Chemokines and chemokine receptors: positioning cells for host defense and immunity.[J]. Annual Review of Immunology, 2014, 32(1):659-702.
 
  
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'''Figure 2. the mechanism of interaction between a chemokine and a chemokine receptor.'''
  
  
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Under the circumstances of inflammation, various kinds of cytokines, including chemokines, are released by the lesions. Guided by the chemokines, cells expressing chemokine receptors move towards the lesions where they can function better.[2] What’s more, different diseases would release different pools of chemokines, which would recruit different effector cells. [https://static.igem.org/mediawiki/2016/2/2d/T--SYSU-MEDICINE--project-diseasse-table-chemokine.pdf See our disease talbe-chemokine]
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1993001 SequenceAndFeatures</partinfo>
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Based on the chemotaxis theory, in order to enhance the homing ability of our marvelous mesenchymal stem cells (MSCs) due to lack of enough chemokine receptors on their cell surface, we, SYSU-MEDICINE, had constructed a series of chemokine receptors that corresponding to different inflammatory diseases as far as possible. Among which, CCL19 is a significant chemokine (CCR7 is its chemokine receptor) in Arthritis.  [https://static.igem.org/mediawiki/2016/2/2d/T--SYSU-MEDICINE--project-diseasse-table-chemokine.pdf See our disease talbe-chemokine]
  
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We acquired this gene from peripheral mononuclear blood cells (PMBCs) and purified it. (Figure 3) Then we constructed it under the control of EF-1α by Gateway technology. (Figure 4)
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===Functional Parameters===
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<partinfo>BBa_K1993001 parameters</partinfo>
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<img src="https://static.igem.org/mediawiki/2016/2/2e/T--SYSU-MEDICINE--CCR7.jpg" style="width:120px"  ></a>
  
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'''Figure 3. Purification of CCR7.'''
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<img src="https://static.igem.org/mediawiki/2016/4/47/T--SYSU-MEDICINE--EF1-a-CCR7.png" style="width:200px"  ></a>
  
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'''Figure 4. EF-1α-CCR7.'''
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We introduced that plasmid into MSCs and tested the expression of CCR7 in MSCs on mRNA and protein level (Figure 5, Figure 6)
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'''Figure 5. Relative mRNA Level of CCR7.'''
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<img src="https://static.igem.org/mediawiki/2016/5/57/T--SYSU-MEDICINE--wbCCR7.jpg" style="width:200px"  ></a>
  
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'''Figure 6. Western Blot of CCR7.'''
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Then, we tested the chemotaxis of engineered MSCs by conducting Transwell assay against CCL19. To our excitement, our engineered MSCs had improved their homing ability with chemokine receptor CCR7(Figure 7,Figure 8).
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<img src="https://static.igem.org/mediawiki/2016/a/ad/T--SYSU-MEDICINE--BBa_K1993001-fig8.jpg" style="width:150px"  ></a>
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'''Figure 7, Figure 8: Tanswell Assay of CCR7.'''
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==MIT_MAHE 2020==
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'''Summary'''
  
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CCR7 is chemokine receptor for CCL19, which is a significant chemokine in Arthritis. It was acquired from peripheral mononuclear blood cells (PMBCs) and purified. It is activated by two different ligands, CCL19 and CCL21, and is responsible for the proper recruitment of lymphocytes and mature dendritic cells to lymphoid tissues.
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==References==
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[1] Allen, Samantha J.; Crown, Susan E.; Handel, Tracy M. (2007-01-01). "Chemokine: receptor structure, interactions, and antagonism". Annual Review of Immunology. 25: 787–820.doi:10.1146/annurev.immunol.24.021605.090529. ISSN 0732-0582. PMID 17291188.
  
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[2] Griffith J W, Sokol C L, Luster A D. Chemokines and chemokine receptors: positioning cells for host defense and immunity.[J]. Annual Review of Immunology, 2014, 32(1):659-702.
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[3] Förster, R., Davalos-Misslitz, A. C., & Rot, A. (2008). CCR7 and its ligands: balancing immunity and tolerance. Nature reviews. Immunology, 8(5), 362–371. https://doi.org/10.1038/nri2297
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[4] Tal, O., Lim, H. Y., Gurevich, I., Milo, I., Shipony, Z., Ng, L. G., Angeli, V., & Shakhar, G. (2011). DC mobilization from the skin requires docking to immobilized CCL21 on lymphatic endothelium and intralymphatic crawling. The Journal of experimental medicine, 208(10), 2141–2153. https://doi.org/10.1084/jem.20102392
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[5] Luster A. D. (2002). The role of chemokines in linking innate and adaptive immunity. Current opinion in immunology, 14(1), 129–135. https://doi.org/10.1016/s0952-7915(01)00308-9
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[6] Link, A., Vogt, T. K., Favre, S., Britschgi, M. R., Acha-Orbea, H., Hinz, B., Cyster, J. G., & Luther, S. A. (2007). Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells. Nature immunology, 8(11), 1255–1265. https://doi.org/10.1038/ni1513"
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1993001 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K1993001 parameters</partinfo>
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<p><em>Inflammmation</em>  During the inlflammation process of multiple diseases,such as IBD and artritis,chemoattraction plays a pivotal role,taking the advantae of chemokines and cytokine in target to its correspongding receptors.<br>
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<em>Chemotaxis</em>  Therefore,stem-cell researchers attempt to transport MSC, a kind of mesenchymal stem cell with outstanding capability of inducing issue regenereation and anti-infllammation,to the injured position for cell therapy.Genetically engineered MSCs with upregulated chemokine receptor can obtain more efficient chemoatrraction ability.</p>
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<p>Chemokine receptors are expressed under the control of constructive promoter EF-1α therefore they could be consistently expressed by MSCs.EF1-α induces the prolonged transcription from plasimid DNA to RNA in Human MSCs,and then the RNA will be translated into proteins and expresses in MSCs, like chemokine receptors are integrated onto the celluar membrane.</p>
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<p>Chemokine receptors are cytokine receptors found on the surface of certain cells that interact with a type of cytokine called a chemokine. They have a 7-transmembrane (7TM) structure and couples to G-protein for signal transduction within a cell [^emphasize] (CXCR5)(Figure 1). Following interaction with their specific chemokine ligands, chemokine receptors trigger a flux inintracellular calcium (Ca2+) ions which initiate the onset of chemotaxis that traffics the cell to a desired location (Figure 2).  </p>
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<li><strong>eGFP</strong></li>
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<li><strong>dTomato</strong></li>
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<h3 id="toc_10">Protein coding sequences</h3>
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<li>CCR7<br></li>
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<li>CXCR1<br></li>
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<li>CXCR4<br></li>
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<li>CCR5<br></li>
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<p>Luciferase-IRES-eGFP<br>
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Luciferase-T2A-dtomato-T2A-hFTH<br>
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CXCR4-IRES-eGFP<br>
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CXCR5-IRES-eGFP<br>
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CXCR4-T2A-Luciferase-IRES-eGFP  </p>
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Latest revision as of 17:58, 23 October 2020


CCR7

Chemokine receptors are receptors found on the surface of certain cells that interact with chemokines. They have a 7-transmembrane (7-TM) structure and couple to G-protein for signal transduction within a cell. [1] (Figure 1) Following interaction with their specific chemokine ligands, chemokine receptors trigger a flux intracellular calcium (Ca2+) ions, initiate chemotaxis and guide the cell to a desired location. (Figure 2)

Figure 1. typical structure of a chemokine receptor.


Figure 2. the mechanism of interaction between a chemokine and a chemokine receptor.


Under the circumstances of inflammation, various kinds of cytokines, including chemokines, are released by the lesions. Guided by the chemokines, cells expressing chemokine receptors move towards the lesions where they can function better.[2] What’s more, different diseases would release different pools of chemokines, which would recruit different effector cells. See our disease talbe-chemokine

Based on the chemotaxis theory, in order to enhance the homing ability of our marvelous mesenchymal stem cells (MSCs) due to lack of enough chemokine receptors on their cell surface, we, SYSU-MEDICINE, had constructed a series of chemokine receptors that corresponding to different inflammatory diseases as far as possible. Among which, CCL19 is a significant chemokine (CCR7 is its chemokine receptor) in Arthritis. See our disease talbe-chemokine

We acquired this gene from peripheral mononuclear blood cells (PMBCs) and purified it. (Figure 3) Then we constructed it under the control of EF-1α by Gateway technology. (Figure 4)

Figure 3. Purification of CCR7.

Figure 4. EF-1α-CCR7.

We introduced that plasmid into MSCs and tested the expression of CCR7 in MSCs on mRNA and protein level (Figure 5, Figure 6)

Figure 5. Relative mRNA Level of CCR7.

Figure 6. Western Blot of CCR7.


Then, we tested the chemotaxis of engineered MSCs by conducting Transwell assay against CCL19. To our excitement, our engineered MSCs had improved their homing ability with chemokine receptor CCR7(Figure 7,Figure 8).

Figure 7, Figure 8: Tanswell Assay of CCR7.

MIT_MAHE 2020

Summary

CCR7 is chemokine receptor for CCL19, which is a significant chemokine in Arthritis. It was acquired from peripheral mononuclear blood cells (PMBCs) and purified. It is activated by two different ligands, CCL19 and CCL21, and is responsible for the proper recruitment of lymphocytes and mature dendritic cells to lymphoid tissues.

References

[1] Allen, Samantha J.; Crown, Susan E.; Handel, Tracy M. (2007-01-01). "Chemokine: receptor structure, interactions, and antagonism". Annual Review of Immunology. 25: 787–820.doi:10.1146/annurev.immunol.24.021605.090529. ISSN 0732-0582. PMID 17291188.

[2] Griffith J W, Sokol C L, Luster A D. Chemokines and chemokine receptors: positioning cells for host defense and immunity.[J]. Annual Review of Immunology, 2014, 32(1):659-702.

[3] Förster, R., Davalos-Misslitz, A. C., & Rot, A. (2008). CCR7 and its ligands: balancing immunity and tolerance. Nature reviews. Immunology, 8(5), 362–371. https://doi.org/10.1038/nri2297

[4] Tal, O., Lim, H. Y., Gurevich, I., Milo, I., Shipony, Z., Ng, L. G., Angeli, V., & Shakhar, G. (2011). DC mobilization from the skin requires docking to immobilized CCL21 on lymphatic endothelium and intralymphatic crawling. The Journal of experimental medicine, 208(10), 2141–2153. https://doi.org/10.1084/jem.20102392

[5] Luster A. D. (2002). The role of chemokines in linking innate and adaptive immunity. Current opinion in immunology, 14(1), 129–135. https://doi.org/10.1016/s0952-7915(01)00308-9

[6] Link, A., Vogt, T. K., Favre, S., Britschgi, M. R., Acha-Orbea, H., Hinz, B., Cyster, J. G., & Luther, S. A. (2007). Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells. Nature immunology, 8(11), 1255–1265. https://doi.org/10.1038/ni1513"


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1111
    Illegal SapI site found at 1017