…visit the local pub and the patron is saying I’ll have a double Felix.
I think this is quite nice.
(applause).
In any case my inspiration came on April 25th 1989 when I went to a seminar given by Paul Brehm
and Paul described the work that Osamu just talked about, about the jelly fish
and described both the fact that the Aequorea if it had calcium would give blue light,
something I had heard of previously and then something I had never heard of before
which was that Aequorea plus calcium plus this wonderful protein called green fluorescent protein gave off green light.
And that you could shine a UV light on GFP and you would get green light.
Now all the time in the previous 10 years, I had been telling people
that the really wonderful thing about working on this small worm that I had been working on was the fact that it was transparent.
Now after you say this for 10 years straight you begin to believe it, that the animal is transparent.
And I told you how much trouble we had looking at how genes were expressed.
So when I heard this I was astonished, I ignored the whole rest of the seminar,
I probably just day dreamed the whole time through about what we were going to do.
The next day I, these are the scribblings from the next day of trying to call up
and find out who was actually doing the work and I found that Douglas Prasher was cloning the CDNA.
We had a little bit of trouble because we got out of touch with each other for several years.
But in 1992 we got back together and Douglas sent us the clone for the CDNA
and this was because I had a very nice graduate student who had just started Ghia Euskirchen
and we were off to try to find out if this would work.
Now I should give you a little background,
people knew by this time that the fluorescent protein didn’t need any other added component.
And that was good because we didn’t want to have to permeabilise the cells or anything to get stuff in.
But the problem was that, I guess its out of order here, I’ve lost the slide, it must be somewhere else in here.
So let me just describe the situation.
The problem is that that primary amino acid backbone had to form a 5 membered ring to it
and no one knew how many enzymes it was going to take to make that.
So there was a gamble and most people bet that it was going to require something else, that it could not work on its own.
Well Ghia got the clone and we talked about how to do it, here’s a picture of Ghia
and within one month of getting the clone this new graduate student had green fluorescing bacteria
as it shows here in her lab notebook.
So we were extremely excited.
But there’s another important point about this, so this seems to say the experiments are pretty easy,
you know just do the experiment, we were very lucky.
And one of the reasons we were lucky is actually up here, it says that use the scope,
she used the scope from 368 engineering terrace and the reason that’s an important note
is she had just gotten her masters in chemical engineering and knew, at Columbia,
knew that there was a really good florescent microscope in her lab, the old lab.
She also knew that the florescence microscope in my lab was a piece of junk.
And that it would not work.
And had we looked down our microscope at her samples, we would have said the experiment didn’t work,
that would have been horrible.
But she had the good sense to realise that our microscope was not very good, it was a rather old microscope.
And so she went off to her old lab, used their microscope and got this wonderful result.
This lead to a problem of sort of how to do science now that we know that we have something exciting to look at
but also know that our equipment is absolutely no good.
I solved this problem first by trying to borrow a departmental microscope until I got kicked off of that.
And then called up the representatives of various companies saying that I wanted to buy a microscope
and would they bring a loner, so I could try it out and wouldn't it be nice if they could leave the loner for a month or so.
And they did and so if you look in our paper you’ll see that there’s a footnote, it’s a rather strange footnote
because it says you can see the florescence on this microscope, that microscope, that one and that one,
I’m thanking all the people that gave, let me use their microscope, in the paper.
In any case we express this in worms, we did a couple of other experiments and then we went off to try to publish this.
And as you know it was published in Science but there were a number of problems, the first problem was the title,
we submitted the paper as "Green florescent protein, a new marker for gene expression",
we were very excited about this as a nice title.
And as you may know the people at Science, the editors look at the papers first
and decide whether they’re worthy enough to go to reviewers.
And so I got a call back saying no it wasn’t worthy enough, because of the title.
And I said what's wrong with the title, they said well every article in Science magazine is new and novel
so you cannot use those words, you have to take the word out of the title.
I don’t like to be told what to do, so the title that the reviewers got was a little bit different from this,
it was "The Aequorea Victoria green florescent protein needs no exogenously added component
to produce a florescent product in prokaryotic and eukaryotic cells."
In other words this is the whole paper.
(applause)
They then, they accepted it and then the copy editor said to me, you know that title, it’s a little bit too long,
could you change it and so I did back to almost the original title which was probably what they wanted in the beginning.
The other thing has to do with the picture here which I was very proud of,
you can actually see this nerve cell growing out with its growth cone, this is in a living animal, I was very excited about this.
And the art editor, cover editor called me and said you know it’s a very nice picture
except there’s one colour that we really try never to use on the cover because it’s very hard to reproduce
and that’s green, can we change it to another colour, and I said no you can’t.
And then the final problem, so these are all the problems you have in trying to publish your work, its really,
the final thing is that we had already given out samples of the C DNA
to quite a number of people and they were already reporting their successes to us.
And we asked people for permission to use their unpublished work in our article and most people readily gave it.
Except for one person who wrote this letter.
"Marty, it is perfectly fine with me if you cite S. Wang’s in my unpublished results in your science paper on GFP,
provided you meet the following conditions.
You make coffee each Saturday morning for the next 2 months, ready by 8.30 AM,
you prepare a special French dinner at the time of the choosing and you empty the garbage nightly for the next month.
This is my wife.
(laughter).
But it was very important because what she did was really the next step in the evolution of GFP,
she was the first person to make a fusion protein, adding GFP to a protein she was interested in.
It was a protein that she was interested in the developing egg, which is this large bit on the right here in this picture,
of drosophila and she wanted to know where that protein went.
And you can see that it is, it’s made in the cells on the left hand side,
it’s transported into the developing egg on the right
and you can see that it’s being deposited at the pole, it’s actually at both poles its being deposited.
And she could actually put this into a mutant that didn’t have the normal gene and this would replace it.
So she knew it was doing the right thing.
And she was able to watch where it was going in the living ovocytes.
So this was a profoundly important thing.
So I think I did all those requirements but she claims I didn’t, so we’re still debating about this, nonetheless it was published.
So what made GFP and what makes GFP very useful, first it can be genetically encoded, we can put it in an organism
and the next line, progeny and all so on all have this as well, we don’t have to do the prep every time.
It’s non invasive, all we have to do is shine blue light on it and we get green light back.
We’re not fixing the animals, we’re not permeabilising them, we can simply shine light on them, which is fairly innocuous.
The molecule is small and monomeric and the advantage there is that it actually can diffuse throughout the cell
if it’s not attached to anything and so you can outline an entire nerve cell and I’ll show you that in a second.
And finally as I’ve said several times now it can be used in living organisms,
so really for the first time we have a dynamic view of life, rather than a static one.
All of this was helped enormously by all the work that Roger Tsien did
and I don’t know if he’s going to talk about any of this at all but just to say quickly
that he changed the emission properties and the excitation properties,
he’s also known for having created all these wonderful colours and for producing these wonderful molecular machines based on fret.
With all these improvements, people started using GFP enormously, there are at least 30,000 papers listed in pub med
that use florescent proteins and I suspect that may be half of what is really there,
because most people don’t use GFP or florescence in the title or the abstract or the key words at all now.
So I’m sure there’s many, many more.
To give you an idea about what this is, some of the people, there’s sort of a gallery, worms, flies,
canola plant up at the top, mice, zebra fish in the middle at the bottom, on the left here is Elba,
the GFP bunny that was produced for Eduardo Kac, the artist who had this as a family pet
but also displayed it to promote discussions about technology and science and the arts and so on.
And you can see on the right hand side a variety of different types of cells expressing GFP.
But as I said the really important thing about GFP is that you can see this in living cells
and I want to give you a couple of demonstrations.
One is this wonderful picture of the dividing drosophila fruit fly embryo
in which what is marked is tubulen so you can see the spindles on all the dividing cells.
And so let me roll the movie and you can see cell division.
You see how wonderfully synchronise it is as more and more nuclei are produced in the cell.
Now this person, Rosalind Silverman Gavrila, is an amazing artist, now she’s not exactly this artist,
but she does have something also called starry night that I want to show you.
And what she’s done is she’s taken GFP and she’s fused it to a peptide that is a nuclear localisation signal.
So whenever there’s a nucleus around GFP gets sucked up into the nucleus.
But when the nuclei break down during the cell divisions in these same embryos, then it diffuses throughout the cell.
And so by changing, doing false colour to have, to show intensity, she came up with this wonderful film.
You can see the nuclei forming and all the GFP going into the nuclei.
And then they all go away in a wave.
Whole embryo fills up and then the next round of nuclei appear.
And then they go away as well.
So obviously there’s been a lot of things that Jeff Lichtman and Josh Sanes
have produced this wonderful way of individually labelling cells with different amounts of coloured,
different colour fluorescent proteins and being able to distinguish a wide variety of cells within the brain.
So there’s been lots of wonderful things that people have done.
One thing, Osamu didn’t really give the story of how difficult it was for him to isolate GFP Aequorean and then find GFP
and I would direct you to his Nobel speech to hear the true story
but it was not an easy job which is why when this film came out with this different way of isolating GFP, it was quite surprising.
So you just take a jelly fish, stick a hypothermic in it and suck out the GFP.
Somehow you then have to do something with chromosomes but it does say green bioluminescence in aequorea
which is what he calls the organism.
And a very rapid response.
And this is the lead in to the movie that proclaims sort of the first human transgenic with GFP and that’s the Hulk,
the movie by Ang Lee, as far as I know nothing like this actually has really occurred.
What I want to do now is talk a little bit about what we’ve done in the lab and what the problems are that we’re starting to face.
So we’ve used GFP in a variety of ways in my lab to study touch sensitivity, we’ve looked to see as shown in the top panel,
if a gene is expressed in the 6 touch sensing cells
and this is an example of one that is in which GFP has diffused all the way through the nerve cells.
The middle panels show by using 2 different colours that we can look at co-localisation
and show that 2 genes are expressed in the same cells.
And finally at the bottom we can show that GFP by being fused to a protein is not localised
or does not diffuse throughout the cell but actually localises to discrete areas within the cell
and this has actually led us to find these mechanosensitive centres that are in the cells.
We have also used this as the basis of a mutagenesis, if you can see a cell you can then take the animal
and mutate it and look for variance.
Things that don’t have the right number of cells in which the nerve cells grow out inappropriately,
maybe in the wrong direction or branch in appropriately.
Or what's really shown here at the bottom is actually having a GFP fusion with a protein
that only goes to the synapses and then asking for mutants that don’t have synapses.
So you can actually look for genes that are important for the connections being made between nerve cells,
there’s lots of different ways depending on basically people’s ingenuity.
The other thing that one can do by labelling cells with GFP is you can separate them out
and the panel in the upper right hand corner shows cells that we’ve taken out of an embryo and grown in culture
and only the touch sensing cells express GFP and they grow out in culture and we can put them through a cell sorter,
isolate them from all the other cells and then ask what RNA’s are over expressed in those cells.
And I’ll mention that in a little bit.
We’ve also used this as a way of labelling cells so that we can electrically record from them
and that’s what's diagrammed in the bottom right hand corner and that's led us to actually produce the first real proof
that we had an animal mechanosensor.
So all of these are different ways that one can use it.
And it really is just the tip of the ice berg since so many other people have made so many other wonderful tools using GFP.
So all of this has led us to having this complex that is needed to sense touch
but having all these components just allows us to have a lot of new questions.
So I’ve labelled a bunch of things in red and I’ll tell you why they’re all in red in a moment as I go through the pictures.
So the question we have is, ok this is what senses touch, what are our questions,
well one of the questions still is how does this animal generate different types of cells,
how does that come about, how do you get different types of cells.
The second one is ok now we have the component, how does it work.
We really don’t know, this is going to take engineers, physicists, chemists
to help us try to understand how this is really working.
All those things in red were proteins that are in some way involved with lipid,
that lipid is somehow very important in regulating the structure.
We haven’t a clue, this is where chemistry is going to become also very important for us.
Fourth question is how can we, you know do we have all the genes yet that are needed for this process.
And finally another question we’re just starting to ask, is ok we’ve been very happy of learning how genes are turned on,
what turns them off?
And so I just want to actually talk about 3 of these very briefly.
How are different cells generated, so we in the past have identified a whole series of genes,
all the things in red are transcription factors, things that turn on genes.
And these are the genes that turn on all those components in these touch cells.
Now interestingly all of these genes are also expressed in another set of cells, the FLP’s.
But none of the touch complex genes are.
So if we do antibody we can see the proteins of the complex in the cells on the left but not on the cells on the right.
So what makes these 2 different cells different?
Well at first we thought we had a wonderful answer.
2 more transcription factors.
And that was it, that was the answer.
So recently we’ve done an experiment.
The worst thing in the world to do, once you have a great idea is to do another experiment.
And so this is the experiment.
We isolated the touch cells, we isolated these other cells, the FLP and we asked what genes are over expressed,
there’s about 200 genes in both of them.
That’s ok, that’s a manageable number of genes.
And then we said well are any in common and 56 of them were in common, that’s ok too because cells should share some genes.
But among the 56 are all of the genes that make up those complexes in the touch cells.
So we are left with a problem, the genes that make the difference between these 2 cells are expressed in both cells
but they seem to be translated into proteins only in one cell.
So the question we have now for the future is how is this happening.
What's happening in terms of this global turning off of protein in one cell as opposed to another?
We have some ideas about how to go about that and I won’t go into that.
I want to go to this next question, do we have all the genes needed for touch, we have this wonderful complex,
we know that it actually does sense touch, this should be enough for us.
But in fact we’re greedy, so I have to explain a little bit of genetics to people.
We started this because the only way we could really get at these genes was to find animals that were touch insensitive,
they could be touch insensitive either because the cells were never made,
that gave us the transcription factors or we could get it because the cells didn’t work
and that’s what we were after and that gave us this complex.
But doing genetics there’s a bit of a problem because there’s some genes,
what if you have 2 versions of the gene, we call this redundancy, you get rid of one, the other one is perfectly fine.
So you don’t see any defects because there’s a replacement already there, so we would never have seen that.
And then there’s this other problem, what if a gene is really important for touch
but it’s also really important in a lot of other cells.
If you get rid of that gene the animal dies.
I can tell you, it’s very hard to tell if an animal is touch sensitive or not if it’s dead.
So we wanted to know could we get other genes.
Now how can one go after getting other genes, well partly was by isolating the cells and looking at the RNAs,
but many of you know that a couple of years ago, just in general, we could change the discussions,
how do we get other genes affecting nerve cells.
And you may remember that a couple of years ago Andy Fire and Craig Mello won the second worm prize, Nobel Prize,
Sydney Brenner, John Sulston and Bob Horvitz got the first and I consider my prize the third worm prize,
for discovering something called RNA interference in which if you give animals double stranded RNA they can,
that will eliminate gene activity.
It’s a very easy way of getting rid of a gene.
And they found that for worms it was really quite wonderful.
At first as the middle panel shows there, you can inject the double stranded RNA.
Then they had a student that was not very good and he missed hitting the right part in the animal but the experiment still worked.
They realised well maybe it wasn’t important where to put it and they could soak the animals in double stranded RNA.
And that’s the panel on the right.
And then someone said well if we can soak them in double stranded RNA and they’ll pick this up,
why not make bacteria that will make the double stranded RNA and have the worms eat it,
so we call that feeding RNAi and that works.
So virtually all of us have in our freezers the collection of all the known c-elegans genes, each in a bacteria,
so if we feed the worms the bacteria it should knock out the genes.
And this is a very useful thing and lots of people have done this, its called feeding RNAi.
There is a problem though, when they’ve looked at approximately 16,000, almost 17,000 genes,
they found 600 genes feeding DNA for those genes, double stranded RNA for these genes,
you could get animals that had some sort of defect, a visible phenotype.
And then there were about 1,100 that were dead.
And then there were all the rest, where there was no effect.
And that means that we don’t know anything about the vast majority of genes, that’s exciting.
So the question is how does that happen, what's the problem here.
So this feeding, this RNAi, feeding RNAi will get rid of genes if the genes are expressed in the muscle.
If it’s in the skin, if it’s in the intestine or if it’s in the germ line, it works great for these things and the animals die.
Unfortunately for somebody interested in how the nervous system works it does not work in nerve cells
and that may be one of the reasons we have so many of these things that are no good.
So the question is why and a couple of years ago Craig Hunter at Harvard discovered a gene called SID-1
and the SID-1 protein is a transporter of double stranded RNA into the cells.
And wouldn't you know it, its there in the muscle and skin and intestine and germ line but it’s not in the nerve cells.
So an obvious solution for us is to make animals that make SID-1 in the nerve cells
and then we can use a mutant background to make sure it’s not expressed anywhere else,
so we can change the pattern up here from this one to that one.
So now it only works in the nerve cells.
So why is this exciting?
It’s exciting for 2 reasons.
We can now go in and look at all those 15,000 genes and ask do they now produce a phenotype,
that’s going to lead us into learning a lot about the nervous system.
To give you one example of that we’ve done, this is a whole series of strains, little busy of a slide,
the white bars are what we started off with and the fact that the animals are 5 out of 5
in all the white bars means there was no RNAi phenotype, we did not make the animals touch insensitive.
But as you go through it and you see the different strains we use, you see at the end of each that there’s a black bar
and that’s our strain.
And now you can see that we’re wiping out the activity.
And we do this for just about every gene in the touch system.
So we finally now have a tool to look at nerve cells.
And so we’re very excited about the idea that we can try to uncover other genes.
And the first, and we can look at those 15,000 but now we can also look at those 1,100 genes that cause the animals to die.
Because now all the calls that they were active in, that were causing the cells to die, don’t express them.
And it’s only in the nerve cells, so we can ask for the first time is there some particular effect in the nerve cell.
And we were very interested in one set of genes for this,
which was a gene that are part of what's called the integrin signalling pathway.
Focal adhesions, attachments of cells, require all these genes diagrammed on the left.
And in worms these same genes are needed in muscle cells to attach the muscles.
Well, because they’re in muscles, when we do RNAi or we have mutations in these genes, the animals die as early embryos,
we don’t know if they’re sensitive to touch or not.
But interestingly all of these components are in the touch sensing cells, when we look at antibody or GFP fusions.
So we desperately want to know what would happen in the animals if we could spare the muscles
and just get rid of this in the touch cells.
And to our great joy we found that virtually all of the genes knock out the effect.
And so this means we have now discovered a completely new pathway needed for touch sensitivity.
We now want to know what its doing and what are the various, how it interacts with our channel and so on.
So we have a lot more genes to go through, we have the whole genome to go through
so we have a brand new toy and we’re very excited.
And the last toy I want to talk about is the problem of turning off transcription,
we often think about development as cells acquiring a gene that turns on things and lets it develop the way it wants.
And in the touch system it’s very much like that, a gene gets turned on, everything keeps on and you make it.
However so we’re looking at one gene called UNC-4 and UNC-4 is made in a bunch of cells.
So there are some cells in the early embryo, the DA cells and then a little bit through larval development,
12 more cells are made and then a little bit later 6 more cells are made and so all these cells,
because GFP is very stable, all these cells are on at the same time.
However we’ve made, you know taking advantage of the ubiquitin system which we’ve heard of in the past,
we have made a variant of GFP that is short lived.
And this is very nice, this version of GFP when the animals hatch, they have the 6 cells
but then as they get a little bit older those 6 cells turn off and we just see the 12 cells.
A little bit later those turn off, we just see 6 cells and then finally in the adults we only see 2 cells
which you can see up there in the picture.
This is a wonderful tool because now we can use genetics and start asking the question what turns these genes off.
And we do it by starting off where they’re off, we mutate the animals, looking for any adults that have more green in them.
And those will be defective in the system shutting off the gene.
We’ve found some, we’re in the middle of cloning them now.
There’s going to be a lot more and we have many more genes that show this temporal expression
and we think that there’s going to be a very good new way to be able to do this.
So let me finish up by saying what I think are some of the lessons of GFP.
First: Science is not an individual occupation, it is cumulative.
The fact that Osamu discovered this wonderful protein that I was able to hear about his work and modify it
and think about how we could use it as a marker.
And of all the wonderful things that then Roger did to change it,
to make it an even more useful thing is an example of what science is in general.
I like to say that in fact all of science is very much, that GFP is a nice metaphor for what we do in science,
we receive light, we receive energy of one type from people and then we give it off in a modified form
that then gets absorbed by other people and so on.
And the whole community of people that have worked on GFP has been inspiring and incredible.
There have been just literally hundreds and hundreds of people that have added value to this wonderful process.
The second thing I want to say is that it is, the students and the post docs that are really the innovators in science.
When we first started giving away GFP I would have the heads of lab call me up
and almost invariably they would say you know I heard you have this marker,
I heard about it from my graduate student or I heard about it from my post doc and that’s where the communication was going.
It wasn’t oh I read about this, it was my graduate student told me about this.
So the communication, the interactions that people have, I hope being fostered by this meeting,
are really the things that are driving science and bring about innovation.
Third, I hope this is an example of how working with jelly fish, how working with c elegans on problems that are very basic,
lead to a number of different insights, certainly a lot of new questions to address.
So basic research is essential, it’s the engine that drives innovation
and it leads to insights in human disease and advances in agriculture and industry.
I personally am somewhat dismayed by what I see too much of as an emphasis on what's called translational research.
That is that we must now forget all about basic research and apply everything that we know to the clinic
and I believe that working on human health is extremely important,
I’m not saying it shouldn’t be but not to the exclusion of basic research.
For the most part we know so little, most of the genes in the human genome encode proteins of unknown function,
we don’t know what they are, we don’t know how they interact.
There’s so much more to learn and there’s so much more to learn in all the organisms that we study.
And this leads me to my last point, which is that all life should be studied, not just model organisms, not just c-elegans,
or not just mice, not just particular human diseases, that there’s so much more out there for us to learn from
and the question that I have is what's going to be the next GFP of the future,
what's going to be the next jellyfish, the next c-elegans.
So thank you very much.
(applause).