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University of Toronto s Peter Roy Discusses His C. Elegans RNAi Work

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At A Glance

Name: Peter Roy

Position: Assistant professor of molecular and medical genetics, University of Toronto

Background: Postdoc, developmental biology, Stanford University — 1999-2002; PhD, Molecular and Medical Genetics, University of Toronto — 1999; BSC, biology, Dalhousie University — 1993

The head of his own lab at the University of Toronto, Peter Roy has published a number of papers, including ones in Nature, Genes and Development, and Molecular Cell. Roy recently took time to discuss his work with RNAi News.

How did you get involved with RNA interference?

It goes back to my graduate work, trying to disrupt the function of a gene called semaphorin in C. elegans. Like Andrew Fire, who really discovered RNAi, I was [trying] antisense. I wasn’t sure what phenotype I was looking for — I was just looking for any phenotype because it was a reverse-genetic approach. I got mixed results — the worms were actually constipated.

That sort of approach sometimes works because in C. elegans, when you inject DNA it forms an extra chromosomal array, and the pieces of DNA you inject can re-arrange in all sorts of ways and sometimes you can get rearrangements such that you may produce double-stranded RNA.

That’s how Andy Fire hit on it, but in an in vitro approach — dirty preps was making antisense work much better than people who were very meticulous and very clean. It turns out that with dirty preps, you were getting both strands, and when you inject the combination of both strands of RNA you get the RNAi effect.

So that was my first encounter with RNAi, although unwittingly at the time.

Then, coming out of my postdoc I did microarray analyses. I did hard-core developmental biology, genetics, [and] molecular biology as a PhD student, and as a postdoc I did a lot of microarray stuff. But at the end of the day, I didn’t feel as satisfied as I did with hard-core developmental biology stuff — being 99.99 percent sure that the gene has a role in this process isn’t as satisfying as knocking it out and observing a phenotype.

So, I pretty much scrapped my postdoctoral approach and Julie Ahringer from University of Cambridge was about to release a genome-wide RNAi library. In coming to this job, I asked my prospective chair: “Can I have all this equipment to automate this, to do high-throughput RNAi screens in the worm?” It was the perfect opportunity: she belonged to a consortium that got this big grant, and in that [the group] wrote up a C. elegans portion for doing high-throughput screens with C. elegans, but they didn’t have anybody to do it.

Here I come, totally independently of them, and say: “This is what I want to do,” and they say: “Yup. Sure. Here’s the money.”

So, we’ve got three robots that are going to help us to these high-throughput RNAi screens, and that’s the story.

Where are you right now then?

My root interest has always been axon guidance. That’s what I tried to do during my PhD and ended up studying genes that control epidermal or epithelial morphogenesis.

So, in the worm, there’s a really poorly characterized process whereby muscle cells extend these membrane extensions called muscle arms to the nerve cord. The end of the muscle arm forms the post-synaptic element of the synapse. There are mutants in the worm where you can move the cord around, and it turns out that the arm can still find the cord, suggesting that motor neurons are secreting a cue that guides the arms to the motor neurons.

In C. elegans, RNAi doesn’t work so well in neurons, but it works great in muscles. So being interested in guidance, I thought that I’m going to study muscle cell migration using RNAi. That’s what we did. … Nobody has looked at these muscle arms before in any detail, [and] we looked at genes that are known to control the migrations of other cell types in the worms. That was the first thing that we did — we built constructs that induce the expression of double-stranded RNA … that target genes that are involved in the migration of other cell types … in bacteria [and] fed the worms the bacteria.

Then, we found out that one gene, the GRB2 homolog in worms called sem-5, has the phenotype that we were looking for, that is the muscle extended ectopic membrane projections — we call them aberrant membrane extensions, or AMEs, for short. This led us to discover that the FGFs are involved in controlling membrane extension from the muscles.

Now, we’re gearing up to do genome-wide RNAi screens in C. elegans to look for other genes that control muscle arm extension.

What about what you’d like to do with RNAi, or see other people do with the technology?

The strength of RNAi in C. elegans is that it’s the only multi-cellular model organism where we can do systematic RNAi screens in a high-throughput fashion. {This] is a resource that produces the RNAi effect, so we can deposit those bacterial clones that express the double-stranded RNA in multi-well plates and deposit our worms into multi-well plates, all robotically.

A human hardly has to touch them. And, we’ve developed a system where a computer will screen the wells for worms. All this is automated, which allows us to go through the genome systematically, look for the phenotypes that we we’re interested in — this is unprecedented. The possibilities are endless after that.

We’ve also developed this high-throughput digital imager that we call HIDI with a company called Elegenics based in Mountain View, California.

We think that we’ll be able to screen a single strain, knock out about 17,000 genes or target them by RNAi, and examine the phenotypic consequences in about 16 days.

This platform, how developed is it?

It’s in its final stages of development.

Will it be commercialized?

Yes. It was developed with me, but I don’t have any commercial interest in it, I just want the robot. I’m not part of the company.

When will it be released?

We should have it in March.

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