Improved Vectors for Gene Silencing in Mammalian Cells

描述（申请人提供）：小干扰rna （Small interfering rna, sirna）因其强大的抑制基因表达的能力而备受关注。引入与基因序列同源的双链RNA （dsRNA）已被证明可以通过称为RNA干扰（RNAi）的过程抑制该基因的表达；该过程通过mRNA抑制或降解使基因沉默，sirna似乎抑制基因表达而不产生非特异性细胞毒性反应。siRNA介导的基因沉默可以通过合成siRNA、体外T7转录系统产生的siRNA和基于质粒的DNA载体转染细胞来实现。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调节。DNA载体的发展，其中siRNA的表达可以随意打开和关闭，在功能研究中具有巨大的价值。这些载体可用于使用siRNA技术创建转基因动物或永久细胞系。通过RNAi开发敲除动物可能更可取，因为它比通过同源重组创建敲除动物花费的时间更少。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，而细胞系通常不被脂质和其他转染试剂转染。这些病毒载体也可以让触发物高效地进入动物体内。利用这些载体，我们将构建一组siRNA载体，用于系统地沉默与癌症有关的基因。本研究开发的载体在功能基因组学、疾病预防和药物设计中具有广泛的应用前景。

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.