In our SynNotch system, we kept the functional sequence of the transmembrane domain from α-CD19 Notch. At the N-terminus, we used anti-HER2 scFV(BBa_K3132005) as the extracellular domain of our synNotch recepter to specifically recognize HER2, the tumor surface antigen we chose as the target, so that our engineered NK cells obtain the ability to response to HER2 on the surface of the cancer cell. We fused Gal4-VP64 to the downstream of the transmembrane domain as the intracellular domain of synNotch. In the presence of tumor marker antigen HER2, Gal4-VP64 fusion proteins will be detached from the cell membrane and function as transcription factors. The released Gal4-VP64 will be located into nuclei and recognize UAS sequence in its corresponding promoter UAS_MinimalCMV (BBa_K3132004) and activate expression of targeted genes.
Selection of tumor cell lines
Before beginning the experiments on synNotch, we first selected two tumor cell lines as cell models. They are breast cancer cell lines SK-BR3 and MCF-7. We measured HER2 expression level of two cell lines by flow cytometry (Figure 1). The results showed that SK-BR3 tumor cells are HER2 high-expressing and MCF-7 tumor cells are HER2 low-expressing. Therefore, we used these two cell lines to test the differentiating effect of synNotch receptors.
Characterization of synNotch receptors and selection of the intracellular transcription factor
Then we tested the response of synNotch receptors with three different transcription factors fused to intracellular segments, hoping we could find a most suitable one with high activity and selectivity. The transcription factors included PIP-VP64(BBa_K3132003), Gal4-VP64(BBa_K3132000), and ZF21-16-VP64(BBa_K3132001). The activity of these three parts can be found on their part pages. Each three synNotch receptor was expressed on the surface of HEK-293 cells. We also transfected reporter plasmids with corresponding promoters regulated by the transcription factors. Then these transfected cells were counted and co-cultured with the two types of tumor cell lines characterized above at the ratio 1:1. Twenty-four hours later, fluorescence intensity was measured to indicate the efficiency of gene activation. All three types of synNotch receptors with transcription factors Gal4-VP64, PIP-VP64 and ZF21-16-VP64 were able respond to HER2 on target cells. The fluorescence intensity detected in co-culture system with SKBR-3 cell line was about twice of that in MCF-7 co-culture system. Moreover, we found that PIP-VP64 had the most effective response, as it had the strongest fluorescence intensity compared with the other two pairs. However, background activity was also observed in all three groups and the leakage in PIP group was the strongest. Considering these two findings, we finally chose Gal4-VP64 as the intracellular domain of anti-HER2 synNotch for it had a relatively strong activity but had a lower background than PIP-VP64.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NotI site found at 1642
- 21Illegal BglII site found at 56
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 985
Illegal NgoMIV site found at 1162
Illegal NgoMIV site found at 1700
Illegal NgoMIV site found at 1748
Illegal NgoMIV site found at 1883
- 1000Illegal SapI.rc site found at 1321