Detection of the efficiency of c⁃Met CAR virus infected T cells by qRT⁃PCR
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    Abstract:

    Objective:To detect the efficiency of c-Met CAR infection in T cells by qRT-PCR using specific primers. Methods:Gene recombination technology was introduced to construct c-Met CAR(GFP)retroviral plasmids. C-Met CAR and c-Met CAR(GFP)viruses were prepared by viral packaging. C-Met CAR-T cells and c-Met CAR-T(GFP)cells were prepared by infection of T cells with virus. The expression of c-Met CAR and c-Met CAR(GFP)virus infected 293T cells was tested by Western blot assay to express exogenous CD3ζ protein. Specific primers were designed to detect the infection efficiency of T cells by qRT-PCR and compared with flow cytometry. Results:c-Met CAR(GFP)plasmid was constructed,and the expression of c-Met CAR(GFP)plasmid on 293T cells was observed by fluorescence microscope. The results showed that c-Met CAR and c-Met CAR(GFP)virus infected 293T cells expressed exogenous CD3ζ protein. The infection efficiency of c-Met CAR and c-Met CAR(GFP)virus were(55.9 ± 2.3)% and(52.1 ± 1.7)% by qRT-PCR. The efficiency of c-Met CAR(GFP)infection was(50.7 ± 3.6)% by flow cytometry. There was no statistical difference between the two methods(P > 0.05). Conclusion:Through the design of specific primers,the qRT-PCR can detect the infection efficiency of c-Met CAR virus on T cells,which is accurate and security,and have value for the clinical application of CAR-T cell therapy.

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黄骁辰,蒯兴旺,杨婷婷,唐 奇,李 涛,季国忠,赵 薇,刘振云,陈 渊,仇镇宁,冯振卿,朱 进.荧光定量PCR检测c⁃Met CAR病毒感染T细胞的效率[J].南京医科大学学报(自然科学版英文版),2018,(7):903-908.

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  • Received:March 30,2018
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  • Online: July 20,2018
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