Journal: Journal of hematology & oncology

Article Title: Engineering a controllable and reversible switch for CAR-based cellular immunotherapies via a genetic code expansion system.

doi: 10.1186/s13045-024-01648-0

Figure Lengend Snippet: Fig. 1 Design and characterization of the reversible CAR protein expression device induced by the genetic code expansion system. A Schematic repre sentation of the genetic code expansion system, including tRNACUA, the orthogonal aminoacyl-tRNA synthetase (aaRS), and a dual fluorescent protein expression cassette. B&C EGFP protein expression in 293T cells incubated with different concentrations of BOCK, as detected by flow cytometry and fluorescence microscopy. D Schematic representation of the BiCAR expression system, in which TAG was inserted into the CAR coding region, individually located between the initiation codon and the CD19 ScFv (the front BiCAR, F-BiCAR), between the CD19 ScFv and the CD8 spacer (the middle BiCAR, M- BiCAR), and between the 4-1BB costimulatory domain and the CD3 zeta domain (the rear BiCAR, R-BiCAR). E&F Western blot analysis showed the expres sion level of the CAR protein in the three BiCAR systems induced by BOCK addition. The data are reported as the means ± SDs (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, not significant (ns) by t tests

Article Snippet: The flow cytometric antibodies for surface markers were added into the staining buffer, including APC-anti-CD69 (BioLegend, USA), APC/Cy7-anti-CD25 (BD Pharmingen, USA), PE/ Cy7-anti-CD8 (BioLegend), PE-anti-CD4 (BD Pharmingen), APC-anti-CD62L (BD Pharmingen), PE-antiCD45RO (BD Pharmingen), PE-anti-PD-1 (BioLegend), PE-anti-CD56 (BioLegend) PE/Cy7-anti-NKG2D (BioLegend), FITC-Streptavidin (BioLegend), PE-Streptavidin (BioLegend), CD19-ADA-biotin (ACRO Biosystems), PE/ Cy7-anti-CTLA-4 (BioLegend), and FITC-anti-(G4S)n (Hycells, China).

Techniques: Expressing, Incubation, Flow Cytometry, Fluorescence, Microscopy, Western Blot