Hung Chuan-Fu, Cheng Tsung-Lin, Wu Ren-Huang, Teng Chiao-Fang, Chang Wen-Tsan
Department of Biochemistry and Molecular Biology, National Cheng Kung University Medical College, No. 1, University Road, Tainan 701, Taiwan, ROC.
Biochem Biophys Res Commun. 2006 Jan 27;339(4):1035-42. doi: 10.1016/j.bbrc.2005.11.113. Epub 2005 Dec 1.
RNA interference (RNAi) is an extremely powerful and widely used gene silencing approach for reverse functional genomics and molecular therapeutics. In mammals, the conserved poly(ADP-ribose) polymerase 2 (PARP-2)/RNase P bidirectional control promoter simultaneously expresses both the PARP-2 protein and RNase P RNA by RNA polymerase II- and III-dependent mechanisms, respectively. To explore this unique bidirectional control system in RNAi-mediated gene silencing strategy, we have constructed two novel bidirectional expression vectors, pbiHsH1 and pbiMmH1, which contained the PARP-2/RNase P bidirectional control promoters from human and mouse, for simultaneous expression of both the protein-coding genes and short hairpin RNAs. Analyses of the dual transcriptional activities indicated that these two bidirectional expression vectors could not only express enhanced green fluorescent protein as a functional reporter but also simultaneously transcribe shLuc for inhibiting the firefly luciferase expression. In addition, to extend its utility for the establishment of inherited stable clones, we have also reconstructed this bidirectional expression system with the blasticidin S deaminase gene, an effective dominant drug resistance selectable marker, and examined both the selection and inhibition efficiencies in drug resistance and gene expression. Moreover, we have further demonstrated that this bidirectional expression system could efficiently co-regulate the functionally important genes, such as overexpression of tumor suppressor protein p53 and inhibition of anti-apoptotic protein Bcl-2 at the same time. In summary, the bidirectional expression vectors, pbiHsH1 and pbiMmH1, should provide a simple, convenient, and efficient novel tool for manipulating the gene function in mammalian cells.
RNA干扰（RNAi）是一种极其强大且广泛应用于反向功能基因组学和分子治疗的基因沉默方法。在哺乳动物中，保守的聚（ADP - 核糖）聚合酶2（PARP - 2）/核糖核酸酶P双向控制启动子分别通过依赖RNA聚合酶II和III的机制同时表达PARP - 2蛋白和核糖核酸酶P RNA。为了在RNAi介导的基因沉默策略中探索这种独特的双向控制系统，我们构建了两种新型双向表达载体pbiHsH1和pbiMmH1，它们包含来自人和小鼠的PARP - 2/核糖核酸酶P双向控制启动子，用于同时表达蛋白质编码基因和短发夹RNA。对双重转录活性的分析表明，这两种双向表达载体不仅可以表达增强型绿色荧光蛋白作为功能报告基因，还可以同时转录shLuc以抑制萤火虫荧光素酶的表达。此外，为了扩展其在建立遗传稳定克隆方面的用途，我们还用杀稻瘟菌素S脱氨酶基因（一种有效的显性耐药选择标记）重建了这个双向表达系统，并检测了耐药性和基因表达方面的选择和抑制效率。而且，我们进一步证明了这个双向表达系统可以有效地共同调节功能重要的基因，例如同时过表达肿瘤抑制蛋白p53和抑制抗凋亡蛋白Bcl - 2。总之，双向表达载体pbiHsH1和pbiMmH1应该为在哺乳动物细胞中操纵基因功能提供一种简单、方便且高效的新型工具。
