Inhibition of Gene Expression from the Human c-erb B Gene Promoter by a Retroviral Vector Expressing Anti-gene RNA

反义基因是转录启动子活性和随后基因表达的有效抑制剂。由于DNA的拷贝数比转录的mRNA的拷贝数少得多，因此靶向启动子区的抗基因寡核苷酸将比以mRNA为靶标的反义寡核苷酸具有优势。这种特性已被用于抑制多种癌基因的表达，以调节肿瘤增殖或病毒活性。我们开发了一种新的逆转录病毒载体，设计用于表达人c-erb Banti-gene RNA并降低细胞中的启动子活性。小鼠成纤维细胞NIH 3 T3细胞用含有与萤火虫荧光素酶报告基因融合的截短的人c-er B B基因启动子的表达构建体稳定转染。向这些细胞中加入靶向人c-erb B基因启动子中26 bp富含嘧啶的元件的c-erb B抗基因逆转录病毒载体，导致细胞荧光素酶活性的剂量依赖性降低。结果表明，荧光素酶活性的降低是由于转录的人c-er B B抗基因RNA对荧光素酶基因转录起始的影响。预期由逆转录病毒载体产生的抗基因RNA抑制适当癌基因的表达并延缓体内肿瘤细胞的生长。

Anti-gene is a potent inhibitor of transcriptional promoter activity and subsequent gene expression. Since the number of copies of DNA is much fewer than those of transcribed mRNA,anti-gene oligonucleotides targeted against the promoter region would have an advantage over antisense oligonucleotides with an mRNA as a target. This property has been exploited to suppress the expression of a variety of oncogenes for regulating tumor proliferation or viral activities. We developed a novel retroviral vector designed to express human c-erb Banti-gene RNA and to reduce the promoter activity in the cells. Mouse fibroblast NIH3T3 cells were stably transfected with an expression construct containing a truncated human c-erb B gene promoter fused to the firefly luciferase reporter gene. Addition to these cells of the c-erb B anti-gene retroviral vector targeted to the 26 bp pyrimidine-rich element in the human c-erb B gene promoter resulted in a dose-dependent decrease in the luciferase activity of the cells. The results suggest that the reduction of the luciferase activity is due to the effect of the transcribed human c-erb B anti-gene RNA on initiation of transcription of the luciferase gene. The anti-gene RNA produced by a retroviral vector is expected to suppress the expression of an appropriate oncogene and to retard the growth of tumor cells in vivo.