Ambion: Your faculty web page on the University of Pennsylvania School of Medicine web site indicates that you are a molecular neurobiologist. Can you elaborate on some of your current projects?

Dr. Eberwine: We are actually doing a variety of things. There are ten people in the laboratory and we have some people applying molecular techniques to the study of diseases – psychiatric and neurological diseases. But most of our basic science work revolves around understanding how a region of the neuron called the dendrite functions. We want to understand how neurons communicate with one another.

We have active programs now in schizophrenia, bipolar disease, fragile X disease and Parkinson’s disease. Pretty broad unfortunately, but it all makes sense in one way – in terms of the connections.

Ambion: The RNA amplification method you developed is now used all over the world for preparing RNA samples for gene array analysis. But this wasn't the application the technique was originally developed for. Could you elaborate on what purpose you originally developed the RNA amplification technique?

Dr. Eberwine: Actually it was originally developed to look at changes in the expression of multiple genes. But it was certainly before microarrays were developed. What we screened were slot blots and cDNA libraries.

Ambion: Could you tell us about the thought processes or experimental steps you took to develop the aRNA amplification technique?

Dr. Eberwine: Well the problem we are faced with as molecular neurobiologists is that the central nervous system is extremely complex. There are an estimated 10¹² neurons in the mammalian brain and they all form connections with one another, with different cells. Trying to isolate RNA from the intact brain, you just get lots of different cell types. Many of the cell types you may not be interested in for a particular disease or for a particular type of scientific question. So we wanted to try to develop a method that would allow us to work with smaller amounts of tissue. And that’s why we worked on the aRNA amplification. We also did single cell PCR. We wanted to apply these techniques to single cell systems. That was our original goal and we succeeded with both the aRNA procedure and PCR procedure.

Ambion: Did you anticipate its widespread use in gene array analysis or analysis of that type?

Dr. Eberwine: I guess at that point I didn’t anticipate it. I certainly hoped that people would use it for looking at coordinate changes in gene expression. But you know, I’ve really given up trying to predict what people will do [laughing]. So certainly that was a hope. I can’t really say I anticipated it, but I certainly hoped that people would.

Ambion: The aRNA amplification method is not the only analytical procedure you've developed.

Dr. Eberwine: No, no, we’ve been very lucky. We’ve actually developed several procedures. One is called In Situ Transcription, which is cDNA synthesis directly on the tissue section, and that has applications for microarray analysis of fixed tissues. We’ve developed single cell RNA transfection protocols. We developed a procedure called IDAT, which is immuno-detection amplified by T7 [polymerase]. It’s a proteomics methodology that has single cell resolution.

Ambion: The IDAT – could you tell me a little bit about that technique and its applications?

Dr. Eberwine: For the IDAT technology, we wanted to not just look at RNA levels at the level of single cells – we wanted to be able to look at protein levels. We have one PNAS paper that’s been published [on the technique] where we used antibodies, [one of which was] an anchor antibody. We would allow antigen to bind to that antibody and then use a second antibody that binds to a distinct region of the antigen. On that [second] antibody, we have a double-stranded DNA that contains a T7 RNA polymerase promoter. So what we’re doing is amplifying that DNA to detect antibody—antigen interaction.

We published a paper on the use of antibodies and single chain Fv phage display libraries for looking at a particular protein. And we’re now in the process of robotizing this. We’re actually up to a hundred proteins from a single cell and we’ll soon be at, I hope, several thousand. The key is that the amplification procedure, the aRNA procedure or cRNA procedure, is linear. And so we can actually get a quantitative number out for the amount of RNA that’s made – which again is a reflection of the abundance of the DNA—antibody complex, or DNA—phage display complex or phage display—protein complex.

Ambion: Proteomics is an increasingly important field of study. How important do you think the role of RNA will be to research within the next 10 years?

Dr. Eberwine: They really go hand in hand. The way in which people should think about cell biology is essentially what was called the central dogma several years ago. DNA gives rise to RNA, RNA gives rise to protein. And while RNA levels are not necessarily a reflection of protein abundances, they tell you the capacity of the cell to make protein. While I think a lot of us really would like to be able to look at proteins, you can’t give up on the RNA either. They’re both intimately involved and associated. So I think [RNA] analysis is only going to become more important as we have more information about proteomics.

Ambion: Do you think the proteomics field will eclipse gene expression techniques?

Dr. Eberwine: Without question, it will not. I think you have to do both hand in hand. What happens in science is the newer technologies sort of catch on and everyone wants to apply them. Then after a couple of years of that, people settle down and think about the questions they actually want to ask. And any of those questions, in terms of functioning of cells, is going to require both RNA and protein analysis. And so I think [proteomics] will not eclipse [gene expression techniques] – it will be a nice adjunct to it.

Ambion: We've recently heard the term transcriptomics. Could you define that?

Dr. Eberwine: Well I think what people are referring to in terms of transcriptome is the RNAs that are made or transcribed from the DNA in a particular cell or a particular tissue. I actually don’t like the phrase. I think we have way too many catchy phrases in biology. It's essentially an expression profile of the cell or a tissue where they've decided to give it a name of transcriptome.

Ambion: Are there any recently developed techniques that you see revolutionizing the molecular biology field in the next 10 years?

Dr. Eberwine: You know that’s a hard one. Certainly one of the places where we have to go is into in vivo genomics and proteomics. We have to do more and more in live cells.

The RNA binding proteins are going to be a major area of investigation because essentially transcription, transport of RNA into the cell soma from the nucleus, translation, [and] sub-cellular localization of the RNA are all controlled by RNA binding proteins. And so I think trying to understand that, and techniques that are associated with understanding that, are going to open up a whole new field of investigation. In fact, I would argue what we should be doing is categorizing genes, not based upon their classification as, say, a ligand-gated ion channel or a g protein-coupled receptor, but rather in terms of the RNA binding proteins that bind to them. Because it's those RNA binding proteins that are going to coordinately regulate the localization and translation of the RNAs. So I think it's just going to be an important area for all of us.

Ambion: What techniques do you think will be most important to molecular biologists such as yourself within the next 10 years?

Dr. Eberwine: Again, I think moving more into the in vivo types of analyses. GFP has been useful, but we need many more molecules like GFP [with] different emission spectra. We also need other types of reagents other than proteins.

Ambion: As we mentioned, you are a professor at the University of Pennsylvania Medical School. Do you still spend time working at the bench?

Dr. Eberwine: Oh, absolutely. It's one of the few things I do well [laughing].

Ambion: About how much time do you spend at the bench?

Dr. Eberwine: Minimally, one day a week. And then hopefully more than that. I wish I could spend more.

Ambion: Do you think it's unusual for someone in your position to spend as much time as you do?

Dr. Eberwine: You know, I hope not. It is one of the problems with science. As you’re promoted up through the academic ranks, what they do is essentially promote you away from the things that got you there. And for me, I love the act of discovery – being the first person to see a result and then discussing it for the first time. It's really a great thrill for me and I’m sure for other people in my position. So I hope this isn’t unique.

Ambion: As a professor at a major medical school, what other responsibilities do you have?

Dr. Eberwine: There’s certainly teaching responsibilities. I teach classes, primarily graduate students. Training of scientists in my laboratory, both pre-doc and post-doc, is a very important activity for me, and one I take very seriously.

I do a lot of teaching outside of the university. I was in charge of the Cold Spring Harbor course on cloning of neural genes for eight years. [I also] teach at the Society for Neuroscience, short courses, and a variety of things. I think the teaching is really an important aspect of what I do. And then, like any academic, there are various other types of committees at the university, NIH review committees and things like that.

Ambion: Right, I suppose grant writing falls in there too.

Dr. Eberwine: Absolutely.

Ambion: So, of your so-called extra curricular activities, which do you find the most rewarding?

Dr. Eberwine: It's really the teaching.

Ambion: How do you balance your many different roles?

Dr. Eberwine: You know, one of the luxuries I have is, as a tenured professor, I can say no to things. And so I do say no to things. I try to do the things that are important to me and to the lab and to the aspects of science that I think are important. I don’t hesitate to say no when I need to.

Ambion: You received a patent for the aRNA amplification technique. What other technologies have you patented?

Dr. Eberwine: I think we have something like 18 patents. We’ve patented procedures for making of 5’ end-biased libraries. We patented the IDAT and APRA procedures. We patented various expression profiles for diseases. We patented a methodology for regulation of translational control with stem-loop sequences, and some various things along those lines.

Ambion: Do you have any advice for academic scientists trying to patent their technologies?

Dr. Eberwine: Number one, they should embrace the idea, because it’s a way of bringing money into their university, and all universities can use additional money. Secondly, it’s a way of helping to control the technology so that it’s developed in a way that you’d like to see. And thirdly, they should be careful of conflict of interests and make sure that they do this with their university’s conflict of interest rules in mind.

Ambion: Do you have an opinion on the patenting of gene sequences and how that affects the dissemination of information?

Dr. Eberwine: I actually don’t think that gene sequence, in and of itself, is something that should be patented. Certainly I think that when you make a discovery of say different abundances of RNAs, or something that’s characteristic of a disease, or know that a particular gene is associated with a disease and you characterize that, I think that’s fine.

Ambion: What's next for James Eberwine?

Dr. Eberwine: I just hope that I can get back in the lab and work more on these various procedures. The types of data that are coming out of the lab, I am really very psyched about. We just need to continue to work and, hopefully, be creative about our analysis.

Ambion: Well we’re all looking forward to seeing what’s coming down the pipeline.

Dr. Eberwine: Thank you.