Second-generation shRNA libraries covering the mouse and human genomes

Abstract

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

Targeting Tankyrase 1 as a therapeutic strategy for BRCA-associated cancer

“…Figure 1S). shRNAs were designed and retrovirus was generated as previously described (Silva et al, 2005). Cells were infected with retroviruses encoding the control shRNA (sh-ctrl) or Tankyrase-1-specific shRNAs (sh-TNK5 and sh-TNK6) and 48 h later seeded in six-well plates and put in selection with puromycin.…”

RNAi-mediated gene silencing in tumour tissue using replication-competent retroviral vectors

“…22,23 The shRNA expression cassette was inserted between the env gene and the 3¢-long terminal repeat of the MLV genome encoded by pAZE-GFP, 24 resulting in pMLV-shRNA (Figure 1a). We used either the env gene of the 4070A strain to provide a broad tropism for human and murine cells (aMLV-shRNA) or that of the ecotropic strain Moloney (MoMLV-shRNA).…”

“…First, the shRNA coding sequence was placed within the miR-30-derived shRNA shuttle backbone. 22,23 This shuttle backbone was previously shown to prevent IFN-responsive gene expression in a retrovirus-based vector system. 15 Although not experimentally tested, it is likely that the miRNA-adapted shRNA used here is therefore more potent and less cytotoxic than conventional shRNA as also demonstrated by other studies.…”

Lentiviral vector‐encoded microRNA‐based shRNA‐mediated gene knockdown of N‐methyl‐D‐aspartate receptors in skin reduces pain

“…The gene knockdown effect of miRNA‐based shRNA whose expression is driven by pol II promoters is 12 times more potent than that of traditional shRNA whose expression is driven by pol III promoters. shRNAs have been reported to be inserted in miRNA context, for example, miR‐30‐based shRNA, which has long chain transcripts containing naturally occurring miRNA and RNAi molecules produced by the endogenous miRNA processing mechanism (Silva et al., 2005; Stegmeier, Hu, Rickles, Hannon, & Elledge, 2005; Zeng et al., 2005). Thus, we used the lentiviral miR‐30‐based shRNA vector system to drive expression of the NR1 subunit in this study.…”

“…Cullen and colleagues produced a template of miRNA‐30 precursor RNA, in which miRNA‐30 was replaced by a target gene sequence and found that it effectively inhibited the expression of the target gene (Zeng, Cai, & Cullen, 2005). Furthermore, miRNA‐30‐based short hairpin RNAs (shRNAs), also called shRNAmir, have been shown to inhibit gene expression 12 times more potently than traditional stem‐loop shRNAs (Silva et al., 2005). To achieve long‐term gene silencing, shRNAs expressed from plasmids or viral vectors can be used instead of small interfering RNAs (siRNAs).…”