We wanted to see whether you could actually intervene in gene expression. Later on, the second part of the development... the further developments which was done in the late 1990s we developed improved systems of specificity and high affinity. I'll come to that in the moment but first I want to talk about the first application gene... intervening gene regulation. So by this time I thought it would be ripe to... to actually take a real example and use the zinc finger... not just to do in-vitro binding DNA sequence, but to intervene. Now in the lab was Terry Rabbits who worked in cancer biology and one day at the lab talks which we had every October, one of his post docs, a man called Isidro Sánchez-García from Spain talked about a... onco-gene, a cancer gene which was created by chromosome translocation which gives rise to leukaemia and the way it's done, is chromosome nine and chromosome 22 interchange the tips of their chromosomes so therefore you make new sequences, new combinations and what happens is that the... a gene called BCR and a gene called ABL which is a tyrosine-kinase... some of the exons of BCR get fused to the axons of ABL so you have a new kind of protein, it's an onco-gene, and it produces in one leukaemia it's the... the junction is such it produces a 190 kilodalton onco-gene protein which is the onco-gene... sorry the onco-protein and another one is 210, these are different ways of fusing the... so it's a family and they all give rise to different kinds of leukaemia. They recognise, one is chronic myeloid leukaemia the other is... BLL it's called, so what Terry was trying to do, he wanted to make antibodies to the protein, because you see the protein, the onco-protein will have a new piece of protein sequence in it. And so, they talked about this and I thought, my god, this will be a very good target for the zinc finger. If we could get a zinc finger to bind to this new DNA sequence with... and which could distinguish this new sequence from the parent sequences, from the bit of BCR and a bit of ABL, they were fused and so... now the reason I chose this, because we did this in-vitro was because there was already a biological assay.
And what Terry Rabbits had done was to transplant the C-DNA of the onco-genes into a cell line called BEV3 developed by David Baltimore which depends for existence, for its continuation I should say, on a cytokine, IL3-interleukin-3 and so what... so I heard from the talk this was copied from Terry really that they were going to put in the antibody and they would then block the onco-gene, cell would then... it wouldn't interact with the parent sequences, interact with the onco-protein and then the... then you'd be able to tell it was no longer oncogenic, it wouldn't be dependent. See an onco-gene once you transplant an onco-gene to a cell it becomes independent of growth factor and they demonstrated that. So we recruited... we borrowed Isidro Sánchez-García from Terry and we had his constructs so we developed a zinc finger construct, three fingers, we used three fingers because the majority of zinc fingers were... that we knew at the time were three... in three fingers at a time and that seemed to... we already knew it was nano-molar binding in-vitro experiments, so we made three fingers to target this junction, the junction between the BCL and ABL genes but we had to discriminate it from the sequence of BCL and ABL itself and we did this and we did two things. One in we showed that making a zinc finger peptide blocked the transcription of the onco-gene and restored the dependence on growth factor and you could see that the... once you've blocked the dependence on growth factor the cells just died by apoptosis, and you could see... we managed... we saw that 90% of the cells into which we'd managed to get the DNA were dead and we also had put a tag on, we made this three finger peptide, put a nuclear localisation tag, nucleaisation signal to go to the nucleus and an epitope tag so we could use immunofluorescence to follow it. This paper was published in Nature in 1994. Now in the same paper, we published an experiment along the way which was not to block a gene, but to switch on a gene and we did this with a cell line and a plasma. This was using a reporter gene, this is sort of standard molecular biology, chloramphenicol transferase gene, CAT gene, and that was put into a report and we made, we put this gene with a nine-base pair sequence which was the target for our zinc fingers construct. And we fused our zinc finger to an activation domain and put it into this... plasma into the cell line. And it switched on the reporter gene. So we had shown that you could intervene in gene expression both in inhibiting a gene expression at the same time activating the gene.
[Q] How do you get the protein in?
In? You do it by transfection, you put up the DNA level so you don't have... but you see you don't have, they weren't very highly efficient in those days, we'd only get about 30 to 40% efficiency. Since then the transfection have now... nowadays we get 70% in and what we did of course was to... because we had immunofluorescence we could target the cells in which the... we could target the cells in which the finger had gone in. We had the epitope.
