Kudo Toshiyuki, Sutou Shizuyo
Laboratory of Functional Genomics, Department of Biological Pharmacy, School of Pharmacy, Shujitsu University, Okayama, Japan.
J Reprod Dev. 2005 Jun;51(3):411-7. doi: 10.1262/jrd.16094. Epub 2005 Apr 5.
Gene silencing with short interfering RNA (siRNA) expression vectors is a powerful method for the analysis of gene functions. For the expression of siRNA in mammalian cells, mammalian U6 small nuclear RNA (snRNA) promoters are widely used. However, the mammalian U6 promoter might not function well in other species. In this study, we cloned four putative chicken U6 promoters by PCR and analyzed their functions. First, we screened the chicken genomic database using the human U6 snRNA gene and identified four candidate sequences. The sequences contained some control elements in their promoter regions, but as we could not rule out that they were pseudogenes, we amplified these sequences and used them as promoters for short hairpin RNA (shRNA) expression. Using the firefly luciferase (Luc) gene as a target, transient expression assays were performed with chicken ovary-derived cells. All four putative chicken U6 promoters exhibited suppressive activity toward Luc, and so could act as a promoter for expression of the snRNA gene in the chicken genome. The promoter activity was not as strong as that of a commercially available siRNA expression vector. This probably reflects artificial sequences between the promoters and synthetic DNA encoding shRNA.
利用小干扰RNA（siRNA）表达载体进行基因沉默是分析基因功能的一种强大方法。为了在哺乳动物细胞中表达siRNA，哺乳动物U6小核RNA（snRNA）启动子被广泛使用。然而，哺乳动物U6启动子在其他物种中可能无法很好地发挥作用。在本研究中，我们通过聚合酶链反应（PCR）克隆了四个假定的鸡U6启动子并分析了它们的功能。首先，我们使用人类U6 snRNA基因筛选鸡基因组数据库，鉴定出四个候选序列。这些序列在其启动子区域包含一些调控元件，但由于我们不能排除它们是假基因的可能性，我们扩增了这些序列并将它们用作短发夹RNA（shRNA）表达的启动子。以萤火虫荧光素酶（Luc）基因为靶标，对鸡卵巢来源的细胞进行瞬时表达分析。所有四个假定的鸡U6启动子均对Luc表现出抑制活性，因此可以作为鸡基因组中snRNA基因表达的启动子。启动子活性不如市售siRNA表达载体的活性强。这可能反映了启动子与编码shRNA的合成DNA之间的人工序列。
