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New RNAi tools enable systematic studies of gene function

An international public-private research team led by scientists at the Broad Institute of MIT and Harvard announced today the construction and availability of an extensive library of molecular reagents to silence most human and mouse genes.

As described in the March 24 issue of Cell, this library consists of small RNA molecules that can switch off genes individually, allowing the user to dissect the genetic underpinnings of normal biology and disease. These RNA-interference (RNAi)-based gene inhibitors are packaged in lentiviruses, enabling their use in virtually all types of human and mouse cells. This work springs from the RNAi Consortium (TRC), a unique collaboration among academic research institutions and leading life science companies with the mission to build comprehensive RNAi libraries and make them available to scientists worldwide.

"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 Broad Institute. "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."

"The RNAi library developed by TRC is a rich resource for biological discovery," said Nir Hacohen, assistant professor at Massachusetts General Hospital and Harvard Medical School, associate member of the Broad Institute and a senior author. "Ongoing studies in my own laboratory to understand how the immune system senses pathogens and appropriately targets its response will be accelerated using these tools."

RNAi gives scientists the ability to turn off an individual gene. Its workhorses are small RNA molecules, each of which i s tailored to match a fragment of a gene's unique DNA. This RNA can then bind to its gene target, rendering it inactive. In order to get the small RNAs into cells, TRC scientists packaged them in lentiviruses. "Across the spectrum of biomedicine, there is a need for tools that can be applied to diverse cell types. This is particularly true in cancer research," said Bill Hahn, assistant professor at Dana-Farber Cancer Institute and Harvard Medical School, associate member of the Broad Institute and a senior author of the study. "For TRC's library, lentiviral delivery is an especially effective means to meet this need."

The parallel analysis of thousands of genes using RNAi allows researchers to more readily pinpoint the genes that control a biological process. Therefore, TRC developed the high-throughput techniques and quality-control measures required for such genome-scale studies. "It is a distinct challenge to achieve consistent and cost-effective RNAi methods and we placed a strong emphasis on this part of the process," said David Sabatini, member of Whitehead Institute for Biomedical Research, assistant professor at Massachusetts Institute of Technology, associate member of the Broad Institute and a senior author. "In the quest to develop comprehensive tools for gene discovery in mice and humans, this technology will be a key piece in the puzzle."

To evaluate the RNAi library's performance, the scientists sampled a subset that targets approximately 1,000 human genes. They systematically inactivated these genes in a human cancer cell line to identify ones that regulate cell division during malignancy. Automated cellular imaging was used to efficiently identify dividing cells in thousands of samples. This approach uncovered more than 100 previously unknown growth regulators in addition to several known players, confirming the library's sensitivity as a vehicle for gene discovery.

"This critical new tool illustrates the requirement for acad emic and industry partnerships to drive scientific innovation," said Eric Lander, director of the Broad Institute and a senior author. "The importance of putting these reagents in the public domain will be demonstrated by the many important biomedical discoveries that will stem from them."


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Source:Broad Institute of MIT and Harvard


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