Development of a species-specific RNA polymerase I-based shRNA expression vector

Abstract

RNA interference (RNAi) can be induced in vitro either by application of synthetic short interfering RNAs (siRNAs), or by intracellular expression of siRNAs or short hairpin RNAs (shRNAs) from transfected vectors. The most widely used promoters for siRNA/shRNA expression are based on polymerase III (Pol III)-dependent transcription. We developed an alternative vector for siRNA/shRNA expression, using a mouse RNA polymerase I (Pol I) promoter. Pol I-dependent transcription serves in cells for production of ribosomal RNA (rRNA), and as such, is ubiquitously and stably active in different cell types. As Pol I-dependent transcription is highly species-specific, Pol I-based system provides an important biosafety advantage with respect to silencing of genes with unknown functions.

Intergenic transcripts originating from a subclass of ribosomal DNA repeats silence ribosomal RNA genes in trans

“…( A ) Scheme of pE3SP‐mrDNA−232/−43 used to express ectopic pRNA. The vector pE3SP contains 5′‐terminal rDNA sequences (−639/−1) including three enhancer elements (grey boxes) fused to 3′‐terminal Pol I transcription terminators (Brenz Verca et al , 2007). The rDNA sequences or unrelated sequences from the MCS of pBluescript SK+ were inserted downstream from the Pol I transcription start site (+1) to yield pE3SP‐mrDNA−232/−43 or pE3SP‐MCS.…”

“…Reporter transcripts were monitored by RT–qPCR using primers that amplify pUC sequences present between the promoter and the terminators. pE3SP‐mrDNA−232/−43, rDNA sequences from −232 to −43 were inserted into pE3SP (Brenz Verca et al , 2007). To synthesize pRNA in vitro , pT7mr−205/−1 was used, a pTOPO ® vector containing mouse rDNA sequences from −205 to −1 fused to the T7 bacteriophage promoter.…”

Recovery of infectious type Asia1 foot-and-mouth disease virus from suckling mice directly inoculated with an RNA polymerase I/II-driven unidirectional transcription plasmid

“…Our results showed that in case of the combined application of polymerase I and II, the reverse genetics system could rescue a RNA virus that has a genome of ∼8500 nucleotides (including poly-(C) and poly-(A) tracts). The species specificity of the RNA pol I promoter limited their utility to cells from different species (Brenz Verca et al, 2007;Martin et al, 2006). By the combined application of CMV promoter without species specificity, an additional rescue method might be applicable to more kinds of virus in more model cell types or more species of animal.…”

RNA interference for improving the outcome of islet transplantation

“…To increase the activity of U6 or H1 promoter, hybrid promoters composed of CMV enhancer and U6 or H1 promoter were constructed for shRNA expression [133–135]. Polymerase I promoter has also been reported for shRNA expression [136], however, the use of pol I promoter is still not well investigated.…”