New RNA libraries can selectively inactivate h...( e involved in human disease or to be k...)

The library of genes was integrated into a retroviral vector that was capable of shuttling the genes into other cell types. The researchers 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 for a short hairpin RNA, researchers using the library to screen for genes affecting a specific cellular process can identify which RNA molecule among the thousands in the library is switching off the activity of a particular gene.

But the retroviral vectors used for shuttling the short hairpin RNAs into cells only went so far. They were not efficient for getting genetic short hairpin RNAs into all cell types. That's where an innovative technique developed by Elledge and his colleagues came in handy. This technique, called "mating-assisted genetically integrated cloning" (MAGIC), greatly assisted the transfer of the short hairpin RNA library into all cell types via bacterial mating.

In order to validate that the library worked in human cells, the researchers 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 Elledge. "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, said the researchers. They emphasized that the current and future libraries will be made available to the research community at a nominal cost through Open Biosystems, Inc
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Contact: Jim Keeley
keeleyj@hhmi.org
301-215-8858
Howard Hughes Medical Institute
24-Mar-2004