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Tools & Techniques: Cost-Effective Vector-Based shRNA Expression, Conditional RNAi in Zebrafish, and More


A team of researchers from the Beijing Institute of Radiation Medicine has published details about a new vector-based shRNA expression system for RNAi that promises to be more cost effective than other approaches

According to the report, which appeared in PLOS One, existing vector-based shRNA-expression strategies, while capable of inducing RNAi in viable cells, tend to be either “error prone or cost prohibitive.”

To overcome these limitations, the scientists created a system that uses either one long oligonucleotide or two short ones at “half the cost of conventional shRNA construction methods” and with a greater than 95 percent cloning success rate.

The new system uses the H1 RNA polymerase III promoter, which does not have the toxicity issues of U6 and which has a “well-defined transcription start site proven to be more flexible than the U6 promoter with regard to +1 sequence changes,” they wrote.

Additionally, “proper isocaudomers were used for easier cascade connected shRNAs construction,” and the CMV-emGFP cassette was used to track shRNA transfection. “This cassette could be replaced easily by other therapeutic genes as a means of over-expressing one gene while concomitantly knocking down another gene.”

To test the system, the researchers used the shRNA method to successfully suppress the hepatitis B virus antigens HBsAg and HBeAg without any detectable shRNA-related interferon responses both in vitro and in vivo.

“The method described here provides an inexpensive and powerful new tool with the potential of down-regulating gene expression that can be applied to a variety of biological systems, including treatment of various diseases,” the researchers concluded.

A group of investigators from the National Chiao Tung University reported on a new, freely available resource for identifying siRNA-mediated mechanisms in human transcribed pseudogenes.

Pseudogenes are genomic DNA sequences homologous to functional genes yet are not translated into proteins, and while they are typically considered the “structurally defective non-functional copies of protein-coding genes, the human genome comprises more numbers of pseudogenes than corresponding functional genes,” the team wrote in Database. At the same time, genome-wide studies have identified actively transcribed pseudogenes with functional potential.

In flies and mice, pseudogene transcripts can be processed into siRNAs that regulate protein-coding genes via RNAi. To see if such a regulatory mechanism is present in humans, the researchers constructed a database, called pseudoMap, that pre-processes the raw public microarray data and deep sequencing data into gene expression profiles for both actively transcribed pseudogenes and their cognate genes and small RNA profiles for pseudogene-derived endogenous siRNAs.

Using the resource, the team found that actively transcribed pseudogenes can act as targets for microRNAs that actually regulate the parental gene.

Unlike the handful of other pseudogene databases that have been previously constructed, pseudoMap provides the gene-expression profiles of an actively transcribed pseudogene and its cognate gene in various experimental conditions, and it curates the pseudogene-derived endogenous siRNAs, the researchers added.

A group of University of California, San Francisco, researchers has reported on the development of a method for conditional RNAi gene silencing in zebrafish, a model organism for which such technology is largely unavailable.

The team designed shRNAs in the microRNA-30 backbone, which has been shown to mimic natural miRNA primary transcripts and potentially be more effective than traditional shRNAs, and reported in Genetics “stable RNAi-mediated gene silencing” in zebrafish employing the yeast Gal4-UAS system.

With the system, the investigators uncovered, at single-cell resolution, the role of atypical protein kinase C-lambda in regulating neural progenitor/stem cell division, and showed effective silencing of the one-eyed-pinhead and no-tail/brachyury genes.

With their approach, they also demonstrated stable integration and germ-line transmission of the shRNAs for atypical protein kinase C-lambda, the expressivity of which is controllable by the strength and expression of Gal4, they wrote.

The study, which is the first to show stable and conditional RNAi-mediated gene silencing employing the Gal4-UAS system in zebrafish, may expand the utility of the organism for both basic scientific research and for the identification of potential therapeutic targets, the UCSF team concluded.

A group of researchers from Boston University School of Medicine and Nagasaki University has published the results of a study using a novel lentiviral vector to delivery shRNAs to resident alveolar macrophages, which are key immune effector cells in the lung, offering a new tool to study the function of these cells and for possibly treating diseases in which they are implicated.

Using the approach, the team was able to durably knock down NF-kappaB signaling in the cells, which resulted in sustained down-regulation of p65, a component of the pro-inflammatory transcriptional regulator, nuclear factor-kappaB, and a “key participant in lung disease pathogenesis,” they wrote in Molecular Therapy.

Delivery of the RNAi molecules to mice specifically decreased induction of NF-kappaB and downstream neutrophilic chemokines in transduced alveolar macrophages, as well as attenuated lung neutrophilia following stimulation with lipopolysaccharide, the investigators added. “Through concurrent delivery of a novel lentiviral reporter vector, we track in vivo expression of [NF-kappaB] target genes in real time, a critical step towards extending RNAi-based therapy to longstanding lung diseases.”

The system revealed that resident alveolar macrophages persist in the airspaces of mice following the resolution of induced inflammation, allowing the cells to be used as “effective vehicles for prolonged RNAi delivery in disease settings,” they concluded.