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逆转录病毒载体被广泛用作对造血细胞进行基因转移的工具 ,转导方法的改进有赖于应用可快速分析并被高效选择的基因标志。为此 ,我们克隆了增强型绿色荧光蛋白 (EGFP)基因 ,并构建可表达EGFP的逆转录病毒载体LGSN ,通过脂质体转染和交互感染方法建立高滴度的逆转录产病毒细胞 ,用以分析对造血细胞标记EGFP基因的可行性。流式细胞术和荧光显微镜检测发现 ,EGFP病毒转录的GP +envAm12细胞和K5 62细胞均可发出稳定的绿色荧光信号 ,阳性率可分别达 97%和 86% ;聚合酶链反应分析显示LGSN转导细胞内有原病毒的整合。上述结果提示 ,作为新一代选择性标志 ,EGFP是适于研究对造血细胞基因转移与表达的报告基因 ,对促进人类基因治疗的研究有重要意义
Retroviral vectors are widely used as a tool for gene transfer of hematopoietic cells, and improvements in transduction methods rely on the use of genetic markers that can be rapidly analyzed and efficiently selected. To this end, we cloned the enhanced green fluorescent protein (EGFP) gene and constructed retroviral vector LGSN that can express EGFP. By using lipofection and cross-infection, we established high titer retroviral vector To analyze the feasibility of labeling EGFP genes for hematopoietic cells. Flow cytometry and fluorescence microscopy showed that EGFP-infected GP + envAm12 cells and K562 cells could both emit stable green fluorescence signal, the positive rates were 97% and 86%, respectively. PCR analysis showed that LGSN Guide cells within the integration of the original virus. These results suggest that, as a new generation of selectable markers, EGFP is suitable for studying hematopoietic cells gene transfer and expression of the reporter gene, the promotion of human gene therapy research is of great significance