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Norbert Perrimon on RNAi and Creating a Drosophila DsRNA Library

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At a Glance

Name: Norbert Perrimon

Position: Professor of genetics, Harvard Medical School Investigator, Howard Hughes Medical Institute

Background: Postdoc, Case Western Reserve University — 1984-1987; PhD, biochemistry, University of Paris IV — 1983

Norbert Perrimon has been a professor at Harvard Medical School since he finished his postdoctoral work in the late 1980s. During that time, he helped create a library of 21,300 double-stranded RNAs directed against all predicted open reading frames in Drosophila cells. He also, with the help of an NIH grant, launched the Drosophila RNAi Screening Center at the medical school, where high-throughput RNAi screens in fruitfly cells can be conducted.

Perrimon recently took time to speak with RNAi News about his work and what’s happening in the RNAi field.

How did you get started with RNA interference?

Basically, my lab was trying to identify genes involved in the early development of the fly embryo. At the time, we had done a lot of genetic screens, which identified many mutations that are very specific phenotypes of embryonic development.

Our goal was to clone those genes and try to find out what they are good for, and really go from the classic approach, going from mutations to genes — you identify any mutation, [and] because of its very specific phenotype, you have to go back and make your way to clone the genes. That is a very slow process.

Our problem was to actually identify [and] to clone many genes, very fast. So when this technique of RNA interference came up, we started to do injections into fly embryos of double-stranded RNA to try to identify genes which would [yield] the phenotypes that we were interested in.

The problem is that the injections of double-stranded RNA into fly embryos is a pretty slow process. There were also a lot of artifacts, [but] the bottom line was that we were really struggling with the injections of double-stranded RNA into fly embryos. We became very excited when Jack Dixon’s lab showed that if you just put the double-stranded RNA directly on the fly cells, you can actually see the very potent RNAi effect or the degra- dation of the corresponding mRNA.

After that, we basically shifted all the technology that we had … into fly cells as a way to design cell-based assays that mimic the processes that we are interested in in vivo, to reduce it to a tissue culture cell-based assay. This allows us to basically identify the genes very quickly.

For example, we’ve been doing quite a bit of work on the Wingless/Wnt signal transduction pathway. In this case, when you study it in the embryo you basically get what’s called a segment polarity phenotype where all the segmen- tation of the embryo is all messed up. Now, we can study the same pathway in tissue culture cells by using a transcriptional reporter, which responds to the Wingless or Wnt signals by turning on the dual-luciferase reporter.

So, we do a systematic RNAi using probes against double-stranded RNAs which cover the entire set of open reading frames, now we can go on and try to basically identify all the genes systematically in the genome which are required for the transduction of the Wingless or Wnt signals.

What we’ve done, basically, is set up a methodology where we have now 21,000 double-stranded RNAs, which cover all the open-reading frames in the fly genome, and we’re testing them systematically. That will give us a library, a list of genes which are assisting the process that we’re studying, so that we can test the results from the other tissue culture cells and go back in vivo to validate them.

How far along in this testing process are you?

Pretty far. We spent about two years or so develop[ing] the infrastructure — there’s a lot of robotics involved, and we had to acquire all the instrumentation. Now, for about six months or so, we’re just doing screens.

It’s already in use now. That’s why we opened up the [Drosophila RNAi Screening] Center [at Harvard Medical School], which is funded by the NIGMS. In the center, people can come in from the outside and just come and do a screen, basically.

If you are from a smaller university, which doesn’t have the resources to do this, you can just come in and, if your screen is good, we have money to help people to actually conduct the screen.

We have duplicated the technology from my lab to make it available to the community. This technology is pretty sophisticated and quite expensive. So we wanted to make sure that people [from] small labs [that] may have developed a very nice assay, to do a cell-based assay all they need is to have access to a facility to do the screens. They can come in, sending someone for a week or two weeks, and then have completed their screens and can go back to their own lab and characterize the targets they have.

You are working on building an RNAi signature database …

That’s a long-term goal. If we do many, many screens — if we do 50 or 100 RNAi screens — at the end we’re going to have a signature of all the genes in the fly genome where we’re going to be able to annotate them and say: ‘Okay, this gene number 2,002 is positive in this screen, this screen, this screen, but not this one, not this one, not this one.’

So, we’re going to be able to have a signature of every open-reading frame in the genome in functional screens in cell-based assays, whatever the screens are. The screens can be for signal transduction, for cell biology, for muscle fusion — for many types of screens.

[With] this huge database of RNAi signatures, we’ll be able to start looking at the information more globally, meaning basically that we’ll be able to start doing grouping, trying to find all the genes which have a similar RNAi signature. [This] would mean that those genes are part of … the same pathway or they may be components of the same sub-cellular machine.

The long-term goal is to really get into systems biology using RNAi information.

What’s the timeframe for putting something like that together, a complete …

Well, it’s never going to be complete. The question is: When are we going to have enough information to be able to extract some meaningful information? [For that] I would say probably in a year or so. I think we need to do about 50 screens, let’s say. Fifty full-genome screens to really start looking at information globally, and hopefully we’ll be able to process one screen a week.

We’re almost there, but we’re not quite there yet.

All this is with the fly genome. Is there any plan to start looking at other genomes?

Yeah. There are many plans. There are tons of people out there which are trying to set up this kind of technology for mammalian cells. … I’m sure within two years or so, the mammalian field will be where we are right now.

What about what you’re working on? Are there other projects using RNAi?

One thing we’re doing, which is quite exciting — well, I think it’s exciting — is we have a project with a number of different labs to study host-pathogen interactions. So, we’re able to put pathogens on fly cells and we can try to identify the factors in those fly cells which are either promoting or decreasing the viability of prolifer- ation of the pathogens.

It’s really very exciting, because I think we’ll be able to systematically identify all the factors in the cell which are involved in the interactions between pathogens and … cells.

Has that project actually started?

Yes, we’ve started that.

Is there anything else you’re applying the technology to?

Well, the technology can be applied to as many things as you can think about. There are many applications, but mostly [I’m working with] the most interesting one of cell biology and signal transduction, and now pathogens.

What about looking at the way RNAi is being used and where it is going. Can you give a prediction about the technology, looking down the road, say, five years?

I think the one thing that will be very exciting will be double-stranded RNA chips, where you can start to immobilize the double-stranded RNAs on a glass slide and then pour your cells on top of it. We are able to miniaturize the technology, because right now the technology is created for a well plate, but it’s good to be able to bring it up to a much more miniaturized version like on a chip — that would be very exciting.

I think that will be a major improvement, in terms of doing screens, to be able to miniaturize the technology. [In addition to] speed, you can start to combine compounds and double-stranded RNA screens. If you have a double-stranded RNAs or siRNAs chip then you can put your cells or add a compound on all those cells and start to combine the results from chemical genetics and RNAi, basically.