Ambion: For historical reasons can you give some details about the original 1998 study in C. elegans demonstrating RNAi ( Nature 391: 806-11)?

This paper was important as it was the first to demonstrate that double-stranded RNA can act as a trigger for gene silencing. Prior to this paper, Ken Kemphues, while working with his student Sue Guo, had noticed that preparations of antisense or sense RNA could inhibit the par-1 gene in C. elegans. Nobody knew how to interpret that — everyone was calling it antisense. My lab started using the technique and realized that it was not antisense and thus decided to give it a different name because of some of the other properties that we discovered. For instance, Sam Driver, a graduate student in the lab, observed that the interference effect could spread from tissue to tissue in the animal. He was learning how to inject, and we believed you had to inject into the germ line, which was the target for the interference. While I was teaching him to inject, like any beginner he was missing a lot, and I asked him to go ahead and keep those animals and we'd see what happened. The next day he checked, and sure enough the interference had spread into the germ line from the site of injection. We then went back and intentionally missed the germ line and showed that we could get interference that would, in fact, spread throughout the animal. This was remarkably potent interference using very small amounts of RNA. We could dilute the RNA tremendously and still have an effect.

The other thing that was really remarkable was that you could have the interference effect skip a generation and affect the next generation. This observation was really the one that made me decide to have my lab work on it and to call it something different. Clearly, anything that could be inherited and transmitted for more than one full generation was truly remarkable and deserved to be investigated further. Also incredible was the fact that worms could transmit the interference effect via the sperm even. These experiments were done with another graduate student, Alla Grishok, who showed the interference effect could be passed via the egg or the sperm and that the interference effect was transmitted as an extragenic factor and did not require the target locus. We were still not sure at this point if this was siRNA or the effect of some longer double stranded RNA. We presented at a couple of meetings, and at one of these meetings Andrew Fire came up to Sam and I and suggested that it could be double stranded RNA that was initiating the interference effect. So really, Andy deserves the credit for thinking that it was double stranded RNA. We didn't think it was, quite honestly, although I think that Sam had been leaning that way. Together (with Andrew Fire) we were able to test several of our genes, and it became clear that dsRNA caused a much more potent effect.

Ambion: By December 2002, RNAi was named as Science 's "Breakthrough of the Year". Did you anticipate the widespread use of RNAi?

I'd have to say absolutely not. I felt like there was something potentially useful, in that C. elegans had this remarkable response to dsRNA. But back in 1998, there was no reason to think that this would work the same way in other organisms. Although, of course, we speculated that it might and even patented the idea that it might. But I think a huge amount of credit goes to the people who took it into other systems and demonstrated that it could work. For example, Rich Carthew's lab was the first to show that it worked in another organism, flies. Then there was the beautiful work by the Sharp, Zamore, Tuschel group showing that they could get these activities in Drosophila extracts; and obviously Greg Hannon's work. These people deserve a huge amount of credit - and actually have gotten it (laughing). These were the people that developed the siRNA technology. Those were things that I think would have fallen out of the other work eventually, but they jumped right in and demonstrated it before we even understood the mechanism.

So it was a big surprise in retrospect. I can think back to how I felt in 1998, and that was, gee, we have to figure out how this works and see if there is some way we could get it to work in other systems because it is so great. But I never would have expected it, at that time, to be something that you could already do so easily. I thought we would need to study it a lot more and thought it would take a lot longer before it became applicable. It has been an absolutely amazing past few years when you consider all the developments.

Ambion: Has the RNAi phenomenon changed the direction of your research in any way?

It hasn't changed the overall direction, but it has generated a whole new direction. Currently, about half of my lab is pursuing our studies in developmental biology, cell fate determination, and control of cell polarity in C. elegans, while the other half is studying RNAi. So now there are really two synergistic groups in the lab. This has been a big challenge for me in the last couple of years, to really have to start a whole new lab. Until 1998-1999, I only had about three people working on RNAi. It was actually hard to convince some people to work on it. Hiroaki Tobara, who made huge contributions to RNAi with his genetic screens, really wanted to study developmental biology. I almost had to beg him to do his genetic screen, and he really came up with good ways of doing it.

So that is really how it happened. I convinced a few people and had some grad students that were interested. Then the post-docs started to trickle in; a few fairly brave post-docs that didn't feel the field was too competitive came to the lab. And that is where we are now. I'm by no means converting the whole lab to studying RNAi, though you could justify that because it is such an important and exciting field. But I think that having our developmental biology group has really been an asset to the RNAi group because it has turned out that many of the genes involved in RNAi are essential and they function in development. So there is a natural synergy there, and we are very intrigued by the possibility that some of the mysteries that we have been unable to solve on the developmental side may turn out to be related to RNA interference-like mechanisms or micro RNA function. It's exciting.

Ambion: Can you speculate as to why transitive RNAi does not exist in mammalian cells?

First of all, I would say that it is probably premature to say that it does not exist. It probably does not exist in all C. elegans cells as well. It is possible that it might exist primarily in stem cells or in the germ line cells, and it hasn't really been looked for yet. I think it is not only possible, but also likely, that RNA signals are transmitted from cell to cell in vertebrates, and we just don't have a handle yet on how to trigger it. I am intrigued by the work from the Hunter lab on the transitive RNAi gene, sid1, in that there is a human homologue and that there might be something like that happening in vertebrate cells, or at least some types of vertebrate cells. siRNAs may not be the transmitted RNA species. So if you are initiating RNAi with siRNAs, perhaps you will never see a transitive RNAi. I think we still have a lot to learn about how that happens. We may well find that RNA is in fact transferred from cell to cell, and we just haven't figured out the pathway that you need; maybe it is a different RNA species, maybe you have to trigger the RNAi in a different way, maybe RNAi does not work that way, but there are micro RNAs that are transmitted from cell to cell, so there is a lot to learn and certainly the possibilities remain open.

Ambion: Where do you see the future of RNAi heading from a basic research perspective?

There is still so much unsolved that the future right now is unpredictable. We have our hands full with the present. We haven't figured out what the components are of the major complexes that function in the various steps of RNAi. We don't know how RNAi is transported from cell to cell. We don't know exactly how RNAi is triggered in terms of dsRNA recognition and processing. We don't know the effector step in any organism. We don't know what the nuclease is. You could go on and on and on. Yet, I'd have to say that one of the most exciting things about the whole field is all of these different pathways that seem to utilize small RNAs and what mechanisms they play a role in. This whole area of discovery, to broaden our view, remains very exciting to me as a scientist.

Ambion: Who would you cite as your greatest scientific influence?

I've wanted to be a scientist ever since I was a little kid. I guess I would have to choose Darwin because the whole idea of the origin of life and the idea of natural selection was such a big part of my upbringing. My dad was a paleontologist, so as a very young kid I was intrigued by our place in the universe and where the heck we came from. I was very inspired by the mystery of life and how things evolve and will have to give Darwin the credit for getting me going. I don't think I would have thought of natural selection on my own (laughing). One of the other things that really got me, and I don't know exactly who did this work originally, was when the first DNA cloning was done. That was probably the inspiration that made me favor molecular biology and genetics rather than some other science.

Ambion: Just for fun, what is the last book you read?

Actually, I'm currently reading Greg Hannon's book, RNAi.

Ambion: And finally, what is next for Craig Mello?

I'm going to stay with C. elegans as a system. I think it is a great system, and it has a lot to teach us because it is a relatively simple animal, yet it holds so many unsolved mysteries. I'm not ready to say, okay, let's just go tackle this problem in vertebrate cells because I've learned through C. elegans that the complexity of almost any pathway is so great that it is easy to delude yourself into thinking you understand how it works. So my goal would be to continue to really get at these pathways using C. elegans, where we have very powerful genetics, and try to keep at that until we can't learn anything more, and that is a long ways off.