New RNAi tools enable systematic studies of gene function

Unique public-private partnership creates RNAi-based inhibitors for nearly every human and mouse gene, makes them available to all genetic researchers

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 is 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 academic 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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About the RNAi Consortium (TRC)

The RNAi Consortium (TRC) was launched in 2005 to create and validate a comprehensive set of RNAi reagents for the entire scientific community. This public-private collaboration seeks to generate the molecular tools to silence nearly all human and mouse genes in a three-year, $18M project. In the coming year, TRC will expand the existing RNAi toolset to achieve near-complete coverage of the mouse and human genomes, which will pave the way for thorough, systematic studies of normal physiology and disease.

TRC involves principal investigators at six research centers based in the greater Boston area: Nir Hacohen (Massachusetts General Hospital, Harvard Medical School); William Hahn (Dana-Farber Cancer Institute, Harvard Medical School); Eric Lander (Broad Institute); David Root (Broad Institute); David Sabatini (Whitehead Institute for Biomedical Research [WIBR]), Massachusetts Institute of Technology); Sheila Stewart (Washington University, formerly at WIBR); and Brent Stockwell (Columbia University, formerly at WIBR).

TRC also includes five life sciences organizations: Bristol-Myers Squibb, Eli Lilly and Company, Novartis, the research institute Academia Sinica-National Science Council in Taiwan, and Sigma-Aldrich.

Data and library access

The methods for library production and an updated list of targeted genes can be found online (www.broad.mit.edu/genome_bio/trc/rnai.html). The TRC human and mouse lentiviral shRNA libraries are distributed through Sigma-Aldrich (www.sigmaaldrich.com) and Open Biosystems (www.openbiosystems.com).

Moffat J. et al. (2006). A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. doi:10.1016/j.cell.2006.01.040

A complete list of the study's authors and their affiliations:

Jason Moffat1,2,4,10, Dorre A. Grueneberg1,10, Xiaoping Yang1,10, So Young Kim1,3,7, Angela M. Kloepfer1, Gregory Hinkle1,3, Bruno Piqani1, Thomas M. Eisenhaure5, Biao Luo1, Jennifer K. Grenier1, Anne E. Carpenter2,4, Shi Yin Foo6, Sheila A. Stewart8, Brent R. Stockwell9, Nir Hacohen1,5,7,11, William C. Hahn1,3,7,11, Eric S. Lander1,2,4,7,11, David M. Sabatini1,2,4,11, David E. Root1,11*

1Broad Institute of MIT and Harvard
Cambridge, MA 02139, USA
2Whitehead Institute for Biomedical Research
Cambridge, MA 02142, USA
3Department of Medical Oncology and
Center for Cancer Systems Biology
Dana-Farber Cancer Institute
Boston, MA 02115, USA
4Department of Biology
Massachusetts Institute of Technology
Cambridge, MA 02139, USA
5Center for Immunology and Inflammatory Diseases,
6Division of Cardiology
Massachusetts General Hospital
Boston, MA 02114, USA
7Harvard Medical School
Boston, MA 02115, USA
8Department of Cell Biology and Physiology
Washington University School of Medicine
St Louis, MO 63110, USA
9Department of Biological Sciences
Department of Chemistry
Columbia University
New York, NY 10027, USA
10These authors contributed equally to this work.
11These senior authors contributed equally to this work.
*Corresponding author

About the Broad Institute of MIT and Harvard

The Broad Institute of MIT and Harvard was founded in 2003 to bring the power of genomics to biomedicine. It pursues this mission by empowering creative scientists to construct new and robust tools for genomic medicine, to make them accessible to the global scientific community, and to apply them to the understanding and treatment of disease.

The Institute is a research collaboration that involves faculty, professional staff and students from throughout the MIT and Harvard academic and medical communities. It is jointly governed by the two universities.

Organized around Scientific Programs and Scientific Platforms, the unique structure of the Broad Institute enables scientists to collaborate on transformative projects across many scientific and medical disciplines.

For further information about the Broad Institute, go to http://www.broad.mit.edu.


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