Gene Silencing Is Golden

A beginner’s how-to on RNAi screening in mammalian cells

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© BSIP/SCIENCE SOURCEIt used to be that finding genes that are important for cellular processes in mammalian cells required randomly disrupting those genes by chemical mutation or by insertion of a transposon into the genome. But then, about 10 years ago, RNA interference (RNAi) came along. The technique involves designing and deploying short strands of RNA that bind to gene transcripts with complementary sequences and stop their translation. The advent of RNAi made it possible to systematically disrupt nearly every single gene in mammalian cells using high-throughput screens.

“For mammalian systems this has opened up a whole new realm of interrogating gene function,” says Scott Martin, team leader of RNAi screening at the National Center for Advancing Translational Sciences (NCATS) at the National Institutes of Health. RNAi screens have uncovered genes required for many key cellular processes—such as cell proliferation, the maintenance of stem cells, and apoptosis—and for cellular pathways that replicating viruses exploit.

Even though RNAi screening techniques have improved in recent years, with libraries stocked with more RNA probes for each gene that are more effective at knocking down their targets, there are pitfalls. “Off-target effects are the major limitation,” Martin says. You have to take steps to weed out RNAis that silence genes other than the one they’re meant to ...

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