Application of TARP luciferase reporter system in function identification of CAR-T cells / 细胞与分子免疫学杂志
Chinese Journal of Cellular and Molecular Immunology
; (12): 397-403, 2023.
Article
en Zh
| WPRIM
| ID: wpr-981879
Biblioteca responsable:
WPRO
ABSTRACT
Objective To investigate a convenient and quantitative solution to activation levels and functional characterization of CAR-T cells by inserting T cell activity-responsive promoter (TARP) nanoluciferase reporter gene system into a lentiviral plasmid containing the gene encoding the chimeric antigen receptor (CAR). Methods The recombinant plasmid was constructed by using whole gene synthesis and molecular cloning techniques. The lentivirus was packaged and was infected with human primary T lymphocytes. Flow cytometry was used to detected the positive rate of lentivirus-infected T cells. The functional characterization of CAR-T cells was identified by luciferase reporter gene system, Western blot, flow cytometry, and small animal live imaging techniques. Results The results of enzyme digestion identification and the plasmid sequencing showed that the recombinant plasmids were constructed, and flow cytometry displayed the normal preparation of CAR-T cells. This system could dynamically respond to the activation of CAR-T cells by luciferase reporter gene system. The functional assay in vitro confirmed that the system could reflect the exhaustion of CAR-T cells, and the small animal live imaging results demonstrated that the system can be used as a tracer of CAR-T cells in mice. Conclusion TARP nanoluciferase reporter gene system provides a more convenient, sensitive and quantitative method for evaluating CAR-T cells activation level, exhaustion phenotype and tracing.
Texto completo:
1
Base de datos:
WPRIM
Asunto principal:
Linfocitos T
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Inmunoterapia Adoptiva
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Regiones Promotoras Genéticas
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Línea Celular Tumoral
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Receptores Quiméricos de Antígenos
Límite:
Animals
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Humans
Idioma:
Zh
Revista:
Chinese Journal of Cellular and Molecular Immunology
Año:
2023
Tipo del documento:
Article