Levenson V V, Transue E D, Roninson I B
Department of Molecular Genetics, University of Illinois at Chicago, 60607, USA.
Hum Gene Ther. 1998 May 20;9(8):1233-6. doi: 10.1089/hum.1998.9.8-1233.
To facilitate gene delivery into animal cells we developed and characterized a family of single-transcript vectors (STVs) with different selection markers expressed from the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV). Retroviral IRES-STVs (R-IRES-STVs) were assembled using an LNCX backbone (Miller and Rosman, 1989). In all of these constructs, a multiple cloning site (MCS) is located immediately downstream of the single promoter and is followed by the IRES sequence and a selectable marker. This configuration ensures that the MCS-inserted gene will be expressed in selected cells. The selectable markers of these vectors provide resistance to G418, puromycin, hygromycin B, histidinol D, and phleomycin. One STV contains green fluorescent protein (GFP) as a selectable marker, permitting FACS-mediated selection, which may prove useful in gene therapy applications. More than 70% of recipient cells could be infected with R-IRES-STVs after one round of infection. Up to 99% of infected cells expressed the reporter gene (GFP) after selection with an appropriate drug. When ecotropic receptor was delivered via R-IRES-STV into human HT1080 cells, populations of drug-selected cells as well as a majority of individual clones were found to be highly susceptible to infection by ecotropic retroviruses.
为便于将基因导入动物细胞，我们开发并鉴定了一系列单转录本载体（STV），这些载体带有从脑心肌炎病毒（EMCV）的内部核糖体进入位点（IRES）表达的不同选择标记。逆转录病毒IRES-STV（R-IRES-STV）使用LNCX骨架构建（Miller和Rosman，1989）。在所有这些构建体中，多克隆位点（MCS）位于单个启动子的紧下游，随后是IRES序列和一个选择标记。这种结构确保插入MCS的基因将在选定的细胞中表达。这些载体的选择标记赋予对G418、嘌呤霉素、潮霉素B、组氨醇D和博来霉素的抗性。一种STV含有绿色荧光蛋白（GFP）作为选择标记，允许通过荧光激活细胞分选（FACS）介导的选择，这在基因治疗应用中可能很有用。一轮感染后，超过70%的受体细胞可被R-IRES-STV感染。在用适当药物选择后，高达99%的感染细胞表达报告基因（GFP）。当嗜亲性受体通过R-IRES-STV导入人HT1080细胞时，发现经药物选择的细胞群体以及大多数单个克隆对嗜亲性逆转录病毒的感染高度敏感。
