Applications of green fluorescent protein as a marker of retroviral vectors.

E S Kandel, B D Chang, B Schott, A A Shtil, A V Gudkov, I B Roninson
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引用次数: 34

Abstract

The Green Fluorescent Protein (GFP) of Aequorea victoria is used as a vital fluorescent tag for the detection and isolation of genetically modified cells. Several modified variants of GFP were tested as marker genes in retroviral vectors containing different backbones and promoter combinations. Constructs allowing for reliable detection of GFP fluorescence and the expression of a cotransduced gene from a strong promoter were identified. Cells harboring such constructs are detectable by flow cytometry, fluorescence microscopy and multi-well fluorescence reading. GFP expression in transduced cells is stable both in vitro and in vivo, and long-term dynamics of GFP-positive fractions in a mixed population can be used to monitor the biological effects of a cotransduced gene. Selection of cells with the highest GFP fluorescence enriches for multiply infected cells. The use of different GFP variants allows one to monitor simultaneously two cell populations transduced with vectors carrying GFPs that differ in their fluorescence intensity or spectral properties and to identify doubly transduced cells. In addition, transcription of an inducible promoter positioned in the opposite orientation to GFP can be monitored by the inhibition of GFP fluorescence. Thus, GFP provides a useful marker for gene transfer by retroviral vectors and extends the range of applications for retroviral transduction.

绿色荧光蛋白作为逆转录病毒载体标记物的应用。
维多利亚Aequorea victoria的绿色荧光蛋白(Green Fluorescent Protein, GFP)是检测和分离转基因细胞的重要荧光标记。在含有不同主干和启动子组合的逆转录病毒载体中测试了几种GFP修饰变体作为标记基因。构建允许可靠的检测GFP荧光和强启动子共转导基因的表达。含有这种结构的细胞可以通过流式细胞术、荧光显微镜和多孔荧光读数检测到。GFP在转导细胞中的表达在体外和体内都是稳定的,并且在混合群体中GFP阳性组分的长期动态可用于监测共转导基因的生物学效应。选择具有最高GFP荧光的细胞可使感染细胞增殖。使用不同的GFP变体可以同时监测两个细胞群,这些细胞群被携带荧光强度或光谱特性不同的GFP的载体转导,并识别双重转导的细胞。此外,定位于与GFP相反方向的诱导启动子的转录可以通过抑制GFP荧光来监测。因此,GFP为逆转录病毒载体的基因转移提供了一个有用的标记,并扩展了逆转录病毒转导的应用范围。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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