Improved Vectors for Gene Silencing in Mammalian Cells

描述(申请人提供)：小干扰RNA(SiRNAs)因其强大的抑制基因表达的能力而备受关注。双链RNA(DsRNA)的引入，在序列上与基因同源，已被证明通过一种称为RNA干扰(RNAi)的过程来抑制该基因的表达；这一过程在转录后通过抑制或降解mRNA而使基因沉默，siRNA似乎抑制了基因的表达，但不产生非特异性的细胞毒反应。通过将合成的siRNAs、基于T7的体外转录系统产生的siRNAs和基于质粒的DNA载体导入细胞，可以实现siRNA介导的基因沉默。然而，需要改进的载体系统，其中来自双链寡核苷酸的siRNA的表达可以通过开关来调节。可以任意开启和关闭siRNA表达的DNA载体的开发将在功能研究中具有巨大的价值。这些载体可以用来创造转基因动物或使用siRNA技术的永久细胞系。通过RNAi开发基因敲除动物可能更可取，因为它比通过同源重组创建基因敲除动物耗时更少。病毒载体，如逆转录病毒和腺病毒siRNA载体，将有助于将siRNA靶标(触发器)传递到细胞系中，而这些细胞系通常不会被脂类和其他转染剂转染。这些病毒载体还可以将触发物高效地传递到动物体内。使用这些载体，我们将构建一组siRNA载体，用于系统地沉默与癌症相关的基因。在这项研究中开发的载体将在功能基因组学以及识别疾病预防和药物设计的靶点方面有广泛的应用。

DESCRIPTION (provided by applicant): Small interfering RNAs (siRNAs) have gained much attention for their powerful ability to suppress gene expression. Introduction of double-stranded RNA (dsRNA), that are homologous in sequence to a gene, has proven to suppress that gene's expression through a process known as RNA interference (RNAi); this process post-transcriptionally silences a gene through mRNA inhibition or degradation, siRNAs appear to suppress gene expression without producing a non-specific cytotoxic response. The siRNA mediated gene silencing can be achieved by transfection of cells with synthetic siRNAs, siRNAs produced by in vitro T7 based transcription system, and plasmid based DNA vectors. However, improved vector systems are needed, where the expression of siRNA from double-stranded oligonucleotides can be regulated by on and off switches. The development of DNA vectors, where the siRNA expression can be turned on and off at will have tremendous value in functional studies. These vectors can be used to create transgenic animals or permanent cell lines using siRNA technology. Development of knockout animals by RNAi may be more desirable because it is less time consuming than creating knockout animals by homologous recombination. Viral vectors, such as retroviral and adenoviral siRNA vectors would be useful to deliver the siRNA targets (triggers) into the cell lines, which are not normally transfected by lipid and other transfection reagents. These viral vectors would also allow high-efficiency delivery of triggers into animals. Using these vectors, we would construct a panel of siRNA vectors for systematic silencing of genes involved in cancer. The vectors developed during this study would have wide applications in functional genomics as well as identifying targets for disease prevention and drug design.