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New method powerfully boosts efficiency of RNA interference (RNAi) in shutting down genes
Date:2/24/2011

d HHMI investigator Stephen Elledge, Ph.D., of Harvard Medical School, explain how they succeeded in solving this problem. Based on an idea by Hannon, a pioneer in RNAi technology, and using molecular tools developed in the Lowe laboratory, the team designed an assay that tests thousands of short hairpin RNA (shRNA) molecules at a time for their ability to shut down genes of interest in cells and identifies the most potent RNAi triggers.

In a pilot experiment to find the most potent RNAi triggers for nine target genesincluding a few hard-to-suppress cancer genesthe team generated genetic codes for about 20,000 shRNAs, each with the potential to shut down any one of the nine target genes. Each genetic code was then inserted into a retrovirus that was also engineered to carry the target geneor the "sensor"and a gene for a fluorescent protein, called a "marker." This design ensured that when cells growing in dishes were infected with the genetically engineered viruses, both the sensor and the marker would always be co-produced within each cell along with one shRNA.

ShRNAs that were inefficient at triggering RNAi failed to spur the destruction of their target (or sensor) genes' RNA and that of the fluorescent marker. So these cells produced a fluorescent protein whose brightness could easily be detected. But shRNAs that were potent RNAi triggers caused the efficient destruction of the target gene's RNA and that of the fluorescent marker as well. Those host cells therefore lacked all traces of fluorescence.

"All we had to do then was to sort these cells out, pull out each cell's genetic material and sequence the short hairpin RNA," explains graduate student Christof Fellman, who together with post-doctoral fellow Johannes Zuber led these efforts. "This gave us the identity of the RNAi trigger that was most potent in shutting down the target gene."

The team found that for each of the nine genes, only about 2.5% of all possible shRN
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Contact: Hema Bashyam
bashyam@cshl.edu
516-367-6822
Cold Spring Harbor Laboratory
Source:Eurekalert

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