Part:BBa_K4040023
pSV40-UAS-GFP Expression Cassette
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1115
Usage and Biology
The composite part consists of two parts: the promoter UAS-PSV40 (BBa_K4040033), GFP Genera (BBa_E0840), which initiates the expression of coding sequence. When the GAL4-KRAB fragment from IL-6 complex receptor BBa_K4040021 is released, it binds to the promoter UAS-PSV40 to inhibit the expression of CAR- FcRγ and reduce the transformation of macrophages into M1 state to alleviate the inflammatory response.
As shown in Figure 2, synthetic receptors contained the gp130 followed by the TEV, IL-6R followed by the TCS, which is fused to Gal4-KRAB and IL-6R followed by the TCS, which is fused to tTA. The cDNA sequences containing the various fusion constructs were cloned into the pELNS vector with the promoter of CMV. Also the reporter GFP (E0840) was fused to the promoter UAS-pSV40 (BBa_K511003). We used mCherry (BBa_J06504) as the reporter and connected it to the pTET (BBa_K1061013), followed by PGK promoter and CD20. 1e7 cells were co-transfected with the plasmid, and were added with different IL-6 concentration 24h post-transfection. Eighteen hours later, the mCherry and GFP fluorescence intensity was measured (figure. 3).
MCherry fluorescence intensity represents pTET pathway expression, which is used to reflect CAR-MERTK expression in vivo. GFP fluorescence intensity represents the expression of UAS-PSV40 pathway, corresponding to the expression of CAR-γ in vivo. As can be seen from the figure, CAR-Ms change from M1 to M2 when IL-6 concentration is 2.5-12.5ng/mL.
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