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The Spitzer Space Telescope recently captured this "double helix nebula," an unusual formation in the Milky Way galaxy with two interweaving strands, like the helical structure of DNA. Similar key discoveries within our own genetic galaxy can be realized with the new tools announced today by The RNAi Consortium.
Image courtesy of NASA/JPL-Caltech/UCLA

Sophisticated telescopes provide penetrating views into the depths of the universe, but such far-reaching sight has largely eluded genome scientists, who seek to navigate our inner cosmos. In the March 24 issue of Cell, an international team of scientists announces new strides in this quest with the construction of an extensive library of gene inhibitors, which can molecularly silence most human and mouse genes. Based on the method of RNA interference (RNAi), these reagents expand the scientific ability to probe the distant depths of mammalian genomes.

RNAi gives scientists the molecular wherewithal to turn off an individual gene, offering insight into that gene's biological function. But tailoring this method to all genes in the genome, particularly in human and in mouse, would enrich the knowledge of how they operate in concert. This is precisely where the RNAi Consortium (TRC), a unique public-private partnership among leading biomedical research organizations, set its sights. Launched in 2005, TRC embarked on a mission to build comprehensive RNAi libraries and to ensure their broad availability to the scientific community. Now TRC reports its first milestone: a phase-one library targeting roughly 12,000 human genes and 10,000 mouse genes.

"Switching off a single gene through RNAi reveals how that gene functions in a particular biological process. When RNAi's potential is applied to thousands of genes—as it has been in fruit flies and nematodes—it can provide a more complete picture of that process," said David Root, a senior author of the Cell paper and the director of TRC and the RNAi Platform at the Ó³»­´«Ã½. "Thanks to this unique public-private effort, we now have new tools to enable the entire research community to realize the potential of RNAi in the two most important species in biomedicine."

TRC's library is a constellation of small RNAs called "short-hairpin" or "shRNAs," a reference to the molecules' physical shapes. Each shRNA is tailored to match a snippet of a single gene's unique DNA sequence. This close fit prevents reading by the cellular machinery and therefore, "silences" the gene. The shRNAs that comprise the RNAi library collectively target a large portion of the mouse and human genomes and are expected to include nearly all of the two species' genes within the next year.

To exert their suppressive influences, shRNAs must first be imported into cells. However, it is not easy to transport foreign bits of nucleic acid into many important types of human and mouse cells. Therefore, TRC scientists chose to package the gene silencers in lentiviruses, which have a notable penchant for infecting a wide range of cells. Their undiscriminating tastes provide researchers with access to previously intractable cell types, including non-dividing cells, such as neurons, as well as cells cultured directly from embryonic tissues, such as embryonic stem cells.

Employing lentiviruses to transport shRNAs to cells does pose a few challenges. For example, generating them in amounts that can accommodate large-scale, genomic studies requires significant effort. Therefore, the scientists developed streamlined, semi-automated methods that can produce them in quantity as well as quality.

The parallel analysis of thousands of genes using RNAi allows researchers to scrutinize a large portion of the genome at once, to more easily find the genes that control a specific biological process. Known as "arrayed screening," this approach can be used to piece together the complex gene networks that underlie normal and disease physiology. To test the library's performance in this setting, TRC scientists sampled a subset of shRNAs, which target approximately 1,000 human genes and focus largely on the protein kinases and phosphatases encoded in the genome. They systematically inactivated these genes in a human colon cancer cell line to pinpoint the ones that regulate cell division during malignancy. The researchers used high-content imaging, a computerized image analysis and data processing platform, to efficiently capture and measure dividing cells in hundreds of samples.

This approach uncovered more than 100 previously unidentified growth regulators in addition to several known players. For many of these, the researchers verified that the growth effects stemmed from the shRNA-mediated silencing of the appropriate target gene. Four novel candidates (YES1, TIE1, ROCK1 and MET) were selected for additional analyses. When silenced with the appropriate shRNA, three showed a clear correlation between the effects on cell division and the amount of gene silencing. Specifically, a dramatic shift in the fraction of dividing corresponded to high levels of gene suppression.

The scientists also noticed that some shRNAs altered cell growth only in particular cell types, for example in cancer cells, but not in other non-malignant cells. This observation raises the question of whether these target unique, cancer-specific differences, which could be potentially exploited for therapeutic purposes. Taken together, these findings offer the first tantalizing glimpse of the path to biological discovery that can be forged with these new RNAi tools.