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Tools & Techniques: Conditional, Reversible RNAi Silencing in Mice; RNA/DNA Hybrid siRNAs; and More


Looking for an alternative to traditional knockout mouse technology, researchers from Harvard Medical School have created a new and rapid method for conditional and reversible RNAi silencing in transgenic mice.

Current RNAi transgenesis approaches in mice, while effective, have a number of technical issues including risk of off-target gene silencing, interference with endogenous microRNA functions, and physiological complications of cellular toxicity and animal morbidity, the researchers wrote in a paper appearing in Genesis.

To address these issues, the team developed a pair of vectors called RUSH — ROSA26 U6 short hairpin — and CRUSH — conditional RUSH, which can be used to generate conditional single-copy transgenic RNAi mouse strains with "less work and providing substantial time savings compared with established methods," they wrote.

The vectors incorporate a green fluorescent protein gene-trap at the ROSA26 locus coupled to Pol III driven Cre-lox regulated shRNA cassettes, enabling knockdown with Cre-rescue or conditional knockdown, respectively.

"The reversible RUSH vector uses Cre-mediated recombination to eliminate the shRNA, causing target gene reactivation for testing reversion of knockdown phenotypes by conditional-rescue," they explained in the paper. "By contrast, the inducible CRUSH vector shRNA is silent until Cre-mediated deletion of a stop sequence."

The investigators provided data from in vitro and in vivo experiments with the vectors.

Despite the ability of siRNAs to effectively knock down specific genes of interest, issues of immune stimulation continue to hamper their use as therapeutics. To overcome this limitation, scientists from Osaka City University have developed novel RNA/DNA hybrid siRNAs that induce lower interferon responses than conventional ones.

Called hetero siRNAs, or HsiRNAs, the oligos are composed of an RNA guide strand and a DNA passenger strand. To boost the molecule's activity, the researchers introduced a single mismatch base pair and an extended guanine-rich flanking sequence at the 5' end of the DNA passenger strand.

The HsiRNAs were tested in HeLa cells and induced "much lower" interferon responses than standard siRNAs, the team wrote in the Journal of Bioscience and Bioengineering.

Seeing a need for tools to examine post-transcriptional modifications to microRNAs, a team from Stanford University has developed a new mass spectrometry-based tool for intact miRNA analysis.

Technologies exist for miRNA expression profiling, such as quantitative reverse transcription polymerase chain reaction and high-throughput sequencing, but miRNAs' small size and lack of a poly(A) tail in their sequences make their detection and quantification challenging, the scientists wrote in a paper appearing in the Journal of the American Society for Mass Spectrometry.

Current tools also are unable to fully detect post-transcriptional modifications on miRNAs, which may have significant functional consequences in both plants and animals.

"Mass spectrometry has been previously utilized for the analysis of nucleic acids and oligonucleotides … [but] mass spectrometry-based RNA studies used enzymatic or chemical digestion of longer RNA species prior to analysis," the investigators noted. The small size of miRNAs, however, makes them "ideally suited" for intact mass spectrometry analysis using electrospray ionization.

The Stanford group identified and characterized synthetic miRNAs on a chromatographic time scale using a reversed-phase high-performance liquid chromatography coupled with a high-resolution LTQ-Orbitrap-Velos mass spectrometer, according to the paper. "In contrast to PCR and genomic sequencing techniques, this approach has the potential to allow us to systematically study miRNA modifications and identify miRNAs that are modified in tissue."

Following a series of experiments testing their approach, the research team concluded that their method is "robust with respect to linear dynamic range, and is able to resolve miRNAs with high mass accuracy."