RNAi Tools Foster Multi-species Research

Postdoctoral researcher Katharine Sepp never dreamed she would work on mice when she joined Norbert Perrimon’s lab in 2002. At the time, Perrimon—an HMS professor of genetics and an investigator with the Howard Hughes Medical Institute—was pioneering RNA interference techniques in cells from the fruit fly. In fact, he established the Drosophila RNAi Screening Center (DRSC) at HMS that year, making the technology accessible to dozens of fly labs for the first time.

Yet while identifying genes involved with neural outgrowth, Sepp embarked on an odyssey filled with unexpected detours and encounters. She built new tools for the screening center and forged unexpected collaborations with mammalian researchers along the way, working with Christopher A. Walsh’s lab at Beth Israel Deaconess Medical Center to validate in mice a promising candidate gene. Her efforts culminated in a July 4 PLoS Genetics paper that may serve as a bellwether in scientific publishing.

“Studies that incorporate multiple species, highlighting similarities or differences for a particular system, naturally hold a great deal of interest at the editorial level,” explained Wayne Frankel, editor-in-chief of PLoS Genetics.

“Gone are the days when you put siRNAs on cells at one end of a machine and Nature papers came out the other end,” added Drosophila researcher Brian Oliver, a senior investigator at the National Institute of Diabetes and Digestive and Kidney Diseases, who has also begun to collaborate with mammalian labs. “Now you have to show that you’ve found something, which requires some pretty extensive follow-up work, often in multiple species.”

As the novelty of RNAi wears off, its usefulness grows, fostering cross-pollination between seemingly disparate labs. RNAi libraries continue to improve, since researchers keep adding reagents and reducing off-target effects. And cost-effective, efficient Drosophila RNAi screens attract attention from mammalian labs, particularly when they involve primary cells.

Fresh cells extracted directly from an organism hold obvious advantages over cells that have been growing in culture for generations, but it’s hard to keep primary cells alive. Through extensive trial and error, Sepp managed to grow primary neurons from Drosophila embryos long enough to label them and complete a full-genome RNAi screen. In collaboration with Pengyu Hong of Brandeis University, Sepp used computer software to distinguish dozens of mutant phenotypes, from excessive axon branching to aberrant cell cluster sizes.

Sepp presented her preliminary data at an HMS Genetics Department meeting, where the gene Ran GTPase piqued the interest of Walsh lab member Sofia Lizarraga. She knocked down the gene in the cortices of mice, producing a remarkable branch arborization (see image) consistent with the phenotype Sepp had observed in the primary Drosophila neurons. The mutant neurons were unable to migrate from one layer of the cortex to the next.

Lizarraga’s discovery reinforced the importance of Sepp’s screen and vice versa. The collaboration made each researcher more confident in her findings.

“We really enjoyed this collaboration because we are accustomed to having to do things from start to finish in mice, where things go very slowly,” said Walsh, the Bullard professor of neurology at HMS. “But the fly system allows one to discover important genes very quickly and then validate them in mice.”

DRSC director Stephanie Mohr predicts that such combined studies will become more common in the future as the price of Drosophila RNAi screens continues to drop and screen assays grow more sophisticated. A lab can complete a genomewide screen at the NIH-subsidized DRSC in eight weeks for approximately $10,000 to $20,000 or a focused screen using an RNAi sublibrary in two weeks for approximately $3,000. The screening center has several RNAi sublibraries, including one for transcription factors (993 genes) and one for kinases and phosphatases (468 genes).

“If you’re studying a new problem and you want to add a few candidates right away, kinases and phosphatases are a great choice because you’ll know how to fol-low up your hits after validating them,” said Perrimon.

The primary cell assays developed by Sepp and others make the DRSC screens even more appealing by increasing the odds of identifying genes that are relevant in vivo. And given the funding climate and competition for space in journals, there’s a good chance that many mammalian labs will begin projects with fly screens.

“All things being equal, investigators who report on more than one species will always attract our attention,” said Frankel of PLoS Genetics.