Applications of Green Fluorescent Protein as a marker of retroviral vectors
Applications of Green Fluorescent Protein as a marker of retroviral vectors
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DOI：
10.1007/bf02674280
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发表时间：
1997-09-01
期刊：
影响因子：
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通讯作者：
Roninson, IB
中科院分区：
文献类型：
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作者：
Kandel, ES;Chang, BD;Roninson, IB
维多利亚水母的绿色荧光蛋白（GFP）被用作检测和分离转基因细胞的重要荧光标记。在含有不同骨架和启动子组合的逆转录病毒载体中，测试了GFP的几种修饰变体作为标记基因。鉴定了允许可靠检测GFP荧光和从强启动子共转导基因表达的构建体。通过流式细胞术荧光显微术和多孔荧光阅读可检测到携带这种构建体的细胞。GFP在转导细胞中的表达在体外和体内都是稳定的，并且混合群体中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荧光的细胞富集多重感染的细胞。使用不同的GFP变体允许同时监测用携带GFP的载体转导的两个细胞群，所述GFP在其荧光强度或光谱性质上不同，并鉴定双重转导的细胞。此外，可以通过抑制GFP荧光来监测位于与GFP相反方向的诱导型启动子的转录。因此，GFP为逆转录病毒载体的基因转移提供了有用的标记，并扩展了逆转录病毒转导的应用范围。
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 harbouring 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 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 application for retroviral transduction.