RNA Libraries Available to Researchers

Vast libraries of short segments of ribonucleic acid (RNA) that can be used to turn off individual human and mouse genes to study their function are now being made available to laboratories studying human biology and disease. Two independent research groups reported their respective RNA interference (RNAi) libraries in the March 25, 2004, issue of Nature.

To construct a library of mammalian genes for short hairpin RNA molecules, researchers first had to settle on an optimal design for a short-hairpin-RNA molecule. "We tested a lot of different things: for example, the length of the hairpin, the loop structure, the structure of the transcript, and what promoters to use,” said Gregory Hannon of the Cold Spring Laboratory (NY, USA; www.cshl.org) and one of the investigators. "And we arrived at an optimal structure for this phase of the science.”

Once an optimized basic design of the short hairpin RNA molecule was finished, the researchers produced a library of genes for short hairpin RNAs that could target 9,610 human genes and 5,563 mouse genes. The genes chosen were those likely to be involved in human disease or to be key molecular switches in the cell. This library of genes was then integrated into a retroviral vector capable of shuttling the genes into other cell types. The investigators also incorporated a DNA "bar-coding” system, by which each RNA molecule can be tagged with a unique DNA sequence. By determining the sequence of a given bar code, library researchers can identify which RNA molecule among the thousands in the library is switching off the activity of a particular gene.

The retroviral vectors, however, were not efficient for getting genetic short hairpin RNAs into all cell types. A new technique called "mating-assisted genetically integrated cloning” (MAGIC) greatly assisted the transfer of the library into all cell types via bacterial mating.

In order to validate that the library worked in human cells, the investigators tested it in a genetic screen designed to report defects in human proteasome function. The proteasome is a key component of the machinery by which the cell breaks down unwanted proteins. "This was a thorough test of the system because there are a great number of different genes whose loss could interfere with proteasome function,” said Dr. Stephen Elledge, of Harvard Medical School (Boston, MA, USA; www.med.harvard.edu), who worked with Dr. Hannon's group. "We found quite a few genes, and concluded that the library had worked quite efficiently as a screening tool.”

Current efforts are aimed at increasing the number of human genes targeted by the library, say the investigators. They emphasized that the current and future libraries will be made available to the research community at a nominal cost through Open Biosystems, Inc., in Huntsville (AL, USA; www.openbiosystems.com).

"For the first time, this give us the opportunity to do a version of forward genetics in mammalian cells, where we can look at hypomorphic mutations, ranging from mild to severe, and their consequences on phenotypes, on what will eventually evolve to a genome-wide scale,” noted Dr. Hannon. "Thus, these libraries will evolve into an important resource for the research community.” Drs. Hannon and Elledge led the first group developing a library, while Dr. Rene Bernards of The Netherlands Cancer Institute (Amsterdam; www.nki.nl) led a second group.