Part:BBa_K3132104
UAS_MinimalCMV_CAR_IL-2_His tag
This composite part consists of one of our synthetic promoters, UAS_minimalCMV(BBa_K3132100), and downstream gene in our first layer cell, CAR_IL-2. The corresponding SynTF of UAS_minimalCMV is Gal4-VP64 (BBa_K3132000), which can bind to the UAS(4*Gal4 binding sites) and activate the minimalCMV promoter, therefore initiating the expression of the downstream gene. As for the design of CAR, the second generation CAR design, which fused a scFv, a CD8a hinge, a CD8 transmembrane domain, a 4-1BB intracellular domain and a CD3ζ chain in tandem was used in our project (BBa_K3132016) (Fig. 1). We designed two chimeric receptors that contain scFv derived from antibodies that recognize human EGFR and HER2, two tumor surface antigens we chose to target. Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) were chosen considering their safety as clinical targeted drugs. We inset the gene of rIL-2_His tag behind CAR. In this way, after Gal4-VP64 binds to the UAS_minimalCMV promoter, the expression of CAR and IL-2-tag will be activated. CAR will be expressed on the cell surface and directly recognizes the EGFR and HER2 on the tumor cell surface, performing the killing function; while the secretion of IL-2-tag will be induced, serving as an indicator of the medical conditions and a signal molecule to trigger the response of external device cells. Constitutively expressed blue fluorescent protein (BFP) was placed downstream of the inducible CAR transgene to identify transduced NK-92 cells (Fig. 1), which is under the control of a PGK promoter. With the fluorescence intensity we can verify the levels of co-transduction.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 395
Illegal NotI site found at 632 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 271
Illegal NgoMIV site found at 340 - 1000COMPATIBLE WITH RFC[1000]
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