关键词: 信使RNA, 电转, 外周血单个核细胞, 绿色荧光蛋白

Abstract: Objective To establish an electroporation method to transfect mRNA into human PBMCs for the preparation of CDR3-grafted γδ T lymphocytes. Methods linearized plasmids pGEM4Z/EGFP/A64 containing EGFP cDNA fragment by the digestion with restriction endonuclease SpeⅠ were used as templates for EGFP mRNA transcription in vitro. EGFP mRNA was transfected into anti-CD3 antibody-stimulated human PBMC by electroporation instrument under different conditions with 500V and 500?s, 400V and 500?s or 300V and 500?s, respectively. The levels of EGFP expression in the cells electroporated with EGFP mRNA were evaluated through inverted fluorescence microscope and flow cytometry. The survival rates of transfected-cells were measured by trypan blue staining. Results Agarose electrophoresis showed that pGEM4Z/EGFP/A64 plasmids were well linearized by SpeⅠ digestion and EGFP mRNAs were successfully transcribed in vitro. 48 hours after mRNA-transfection, EGFP expression in cells transfected EGFP mRNAs under the condition of 500V and 500?s attained 77% which were significantly higher than those in cells transfected EGFP mRNAs under other conditions. The survival rate of transfected cells under the condition of 500V and 500?s reached up to 85%. Conclusions The electroporation platform of mRNA transfection into human PBMC with parameters of 500V and 500?s is successfully established and could be useful to prepare genetically modified T lymphocytes by mRNA transfection.

Key words: mRNA, electroporation, PBMC, EGFP.