Difference between revisions of "Part:BBa K4421021"
 
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This part is a CAR inducible promoter containing six NFAT-RE sites in a minimal IL-2 promoter to generate CAR response elements, which was placed upstream of the transcription factor Gal4-KRAB.In our engineered NK-92 cells, NFAT was coupled with KRAB activation after CAR stimulation.
 
This part is a CAR inducible promoter containing six NFAT-RE sites in a minimal IL-2 promoter to generate CAR response elements, which was placed upstream of the transcription factor Gal4-KRAB.In our engineered NK-92 cells, NFAT was coupled with KRAB activation after CAR stimulation.
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<div><ul>
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<center>
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  <li style="display: inline-block;"> [[File:NFAT-RE.jpeg|thumb|none|400px|<b></b> Pathway of NFAT and its downstream structure.]] </li>
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</center>
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    </ul></div>
  
 
===Usage and Biology===
 
===Usage and Biology===
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This part can serve as a downstream promotor of CAR recognition in synthetic immunology, especially in constructing CAR-classis cells with unique circus.Together with synnotch,we can really do a lot.  
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This part can serve as a downstream promotor of CAR recognition in synthetic immunology, especially in constructing CAR-classis cells with a unique circus. Together with synnotch, we can do a lot.  
 +
 
 +
===Supplementary Information===
 +
It consists of six NFAT-RE sites. NFAT-RE can bind with NFAT factors, thus activating the expression of the downstream genes, which is original from the IL-2 promoter.
 +
The regulation of the IL-2 gene was revealed comprehensively during the last century. Several factors are required for induction of the IL-2 gene after stimulation, including NFAT1, NF-KB, AP-1, and octamer-binding proteins(Shapiro et al.,1994). It was previously reported that synthetic NFAT promoter could be successfully applied in CAR-T cells (Chinnasamy D et al.,2012). However, in NK-92 cells, a previous study showed that reporter expression and fold induction from the 4xNFAT promoter were very modest (Kulemin, S.V. et al, 2019). So we designed the NFAT-RE with 6xNFAT and the expression test with GFP showed that it did work in our project.
 +
 
  
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===supplementary information===
 
−
It consists of six NFAT-RE sites. NFAT-RE can bind with NFAT factors, thus activating  expression of the downstream genes, which is original from the IL-2 promotor.
 
−
The regulation of IL-2 gene was revealed comprehensively during last century.
 
 
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
 
<partinfo>BBa_K4421021 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4421021 SequenceAndFeatures</partinfo>
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===Reference===
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Shapiro et al.(1994).c-Rel Regulation of IL-2 Gene Expressionism May Be Mediated Through Activation of AP-1.Journal of Experimental Medicine, 184(5):1663-1669.https://doi.org/10.1084/jem.184.5.1663
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Chinnasamy D et al.(2012).Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice.Clinical Cancer Research,2012;18:1672–83.https://doi.org/10.1158/1078-0432.CCR-11-3050
 +
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Kulemzin et al.(2019).Design and analysis of stably integrated reporters for inducible transgene expression in human T cells and CAR NK-
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cell lines.BMC Medical Genomics,12(Suppl 2):44.https://doi.org/10.1186/s12920-019-0489-4
  
  

Latest revision as of 07:18, 16 July 2022


NFAT-RE promotor

This part is a CAR inducible promoter containing six NFAT-RE sites in a minimal IL-2 promoter to generate CAR response elements, which was placed upstream of the transcription factor Gal4-KRAB.In our engineered NK-92 cells, NFAT was coupled with KRAB activation after CAR stimulation.

  • Pathway of NFAT and its downstream structure.

Usage and Biology

This part can serve as a downstream promotor of CAR recognition in synthetic immunology, especially in constructing CAR-classis cells with a unique circus. Together with synnotch, we can do a lot.

Supplementary Information

It consists of six NFAT-RE sites. NFAT-RE can bind with NFAT factors, thus activating the expression of the downstream genes, which is original from the IL-2 promoter. The regulation of the IL-2 gene was revealed comprehensively during the last century. Several factors are required for induction of the IL-2 gene after stimulation, including NFAT1, NF-KB, AP-1, and octamer-binding proteins(Shapiro et al.,1994). It was previously reported that synthetic NFAT promoter could be successfully applied in CAR-T cells (Chinnasamy D et al.,2012). However, in NK-92 cells, a previous study showed that reporter expression and fold induction from the 4xNFAT promoter were very modest (Kulemin, S.V. et al, 2019). So we designed the NFAT-RE with 6xNFAT and the expression test with GFP showed that it did work in our project.


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
    COMPATIBLE WITH RFC[1000]

Reference

Shapiro et al.(1994).c-Rel Regulation of IL-2 Gene Expressionism May Be Mediated Through Activation of AP-1.Journal of Experimental Medicine, 184(5):1663-1669.https://doi.org/10.1084/jem.184.5.1663

Chinnasamy D et al.(2012).Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice.Clinical Cancer Research,2012;18:1672–83.https://doi.org/10.1158/1078-0432.CCR-11-3050

Kulemzin et al.(2019).Design and analysis of stably integrated reporters for inducible transgene expression in human T cells and CAR NK- cell lines.BMC Medical Genomics,12(Suppl 2):44.https://doi.org/10.1186/s12920-019-0489-4