Mobile website
Archive > Volume 30 Issue 2 > 2023,30(2):156-160. DOI:10.3872/j.issn.1007-385X.2023.02.009 Prev Next
Technigue and Method
Construction and functional identification of CD19-CAR vector based on PiggyBac transposable system
- Article
- Figures
- Metrics
- Preview PDF
- Reference
- Related
- Cited by
- Materials
Abstract:
Objective: To develop an electroporation CAR-T cell preparation method based on PiggyBac (PB) transposable system and to characterize its anti-cancer function in vitro. Methods: Healthy human peripheral blood mononuclear cells (PBMCs) were used to obtain T cells. The CD19 gene was cloned into PB plasmid (transposon) by molecular cloning technique. Then, the transposon and transposase plasmid were introduced into activated T cells by electroporation, and the transfection efficiency was measured. Finally, the ability of prepared CAR-T cells to kill human Burkitt's lymphoma Raji cells was evaluated by flow cytometry and luciferase luminescence assay. Results: The transfection efficiency of CD19 CAR-T cells prepared by electroporation was high (>60%) in a dose-dependent manner, and CAR-T cells had a significantly higher cytotoxicity to Raji cells relative to the Pan-T cell group (P<0.05).Conclusion: The developed PB transposable system is feasible for electrotransfection and has the potential for clinical application inCD19 CAR-T cell preparation due to its significant in vitro cancer cell killing ability.
Keywords:
Project Supported:
Clc Number:
