The 1984 Cold Spring Harbor Symposium was an exceptional meeting for me in many ways. The first excitement was because I got to see so many Nobel Laureates in one place, including legends like Jim Watson and Barbara McClintock.
I still remember Jim’s obituary of Ahmad Bukhari, one of the cleverest molecular geneticists of the time, who had died recently of heart attack while jogging in Cold Spring Harbor…Jim was hardly in the expected somber mood. He managed to blurt out statements such as, “…who had ever heard of a scientist from Pakistan?” and immediately realizing it was not the right thing to say added emphatically, “He was really very, very good, as good as you get!” Evidently Watson had forgotten the Nobel winner Abdus Salam, the PhD advisor of his close associate and friend, also a Nobel laureate, Walter Gilbert. Rasika Harshey, once a postdoc with Bukhari, patched up the uncomfortable silence with a marvelous collection of slides of Bukhari (one in which he was all twisted up in telephone cables while trying to talk to two phones simultaneously) and the snowy windows of the lab in winter.
The meeting was also memorable for a compelling set of experiments reported by Howard-Flanders of Yale, and Jim Watson shouting from the back of the hall, “Francis and I considered a four-stranded DNA model thirty years ago…it does not work!” Later work has proved Watson correct, and yet Howard-Flanders was in the right track and would have probably discovered the real mechanism of DNA strand exchange in time were it not for his untimely death a few years later.
It was also the meeting where I saw Frank Stahl towering over Barbara McClintock, the two standing by the bay, a few geese waddling by. As I approached, I heard Frank exclaim, "...but no one understood what you wrote in those days...even now I don't understand them…you meant for no one to understand what you wrote!" McClintock shook her little walking stick in the air, "Franklin, you will never grow up!"
Hardly memorable at that meeting were two talks, one by Oliver Smithies laboratory reporting the first replacement of a gene in a mammalian cell line, and the other by Mario Capecchi’s lab reporting on recombination of introduced DNA in mammalian cells. The mammalian system was considered ‘dirty’ by most geneticists, and one did not “waste clean thoughts on dirty” genetics. Yeast and phage lambda were the chosen ones, mammalian cells and plants were at the edges of attention.
Capecchi, a former student of Jim Watson, who had done some pioneering biochemical work during his PhD in figuring out parts of the genetic code, had been trying to develop a gene knock-out strategy for mammalian cells so he could study the genetic basis of body patterning in mammals. Smithies, who had earlier discovered gel electrophoresis in the early 1950s, was interested in the genetic basis of human diseases. He wanted to knock out certain mouse genes to test if mutations in these genes could cause human diseases. Both of their experiments consisted of taking mammalian cells in culture, micro-injecting or introducing by other methods some DNA, bearing sequences similar to the target genes that they wanted to knock out, and studying what happens.
Their talks were deep into technical details on how long DNA sequence homology must be, whether little variations in the sequence might affect the outcomes, what kind of selectable marker genes are to be used and such technical considerations. These were scouting works, far removed from the elegant model building and testing that yeast and phage recombination geneticists were doing.
My friend Abed Chaudhury, originally from Bangladesh, whom I had met a few months earlier in Eugene, Oregon, had an exciting new result concerning a critical idea in DNA recombination…he was a flurry of ideas, hard to keep up with. In fact I, a late comer into the field, felt a little stupid in his presence; but we had other shared interests, in poetry and an ambition to translate Carl Sagan’s “Cosmos” into Bengali! And it was reassuring that Abed was always gracious. Abed and I dived into the harbor’s bay on a hot and sultry afternoon, but had to get off the water relatively quickly shivering—the water too cold for us used to our subcontinent’s warmth.
The Double strand break repair (DSBR) model was recently proposed by Terry Orr-Weaver, Jack Szostak, Rodney Rothstein and Frank Stahl, and this meeting saw a number of presentations for or against that model. The model was quite heretical, though a variant of sorts was proposed earlier by Mike Resnick. The surprising aspect of the model was the proposition that for DNA recombination both strands of one of the two recombining DNA molecules must be broken…this was thought to be rather unsavory…it left too much opportunity for the DNA strands to get lost in the process--a rather untidy way to accomplish “clean” recombination as observed by the fungal geneticists. So the DSBR model was not quite accepted yet. In this light, it was unusual that both Capecchi’s and Smithies’ groups had been using linear double stranded DNA with two out-ward facing ends (“ends out”) for targeting experiments whereas earlier work with yeast had shown that circles with “ends in” configuration worked nearly a thousand fold better. I remember walking away feeling vaguely interested that both Capecchi and Smithies groups got high frequency recombinants with “ends out” DNA.
Over the next 15 years, a string of papers from the laboratories of Capecchi and Smithies smoothened out most of the creases on gene knockout by homologous recombination in mammalian cells. The use of positive-negative selection was an important key. The use of large flanking DNA homology and the use of embryonic stem cells were others. The technique is now routine, and its use provides the ultimate test of a human gene’s function (confirmed in mouse).
Many interesting observations remain. While it is now relatively certain why “ends in” type of constructs do not work so well (probably because of the existence of a process called ‘non-homologous end joining’ or NHEJ, discovered only in the 1990s, which snares the introduced DNA into random locations in the genome), it is not clear why embryonic stem cells are one of the very few cell types where knock-out recombination can occur at high frequency. Noting that embryonic stem cells lack in some of the very same recombination gene activities that are thought to be important in the process, it is a rather curious observation.
This year’s Nobel Prize in Physiology or Medicine has gone to Capecchi and Smithies, to share with Sir Martin Evans who first isolated embryonic stem cells in mouse and used it for generating mice with knocked out genes.
It is interesting to note that the gene targeting methods were developed by these scientists merely as tools to investigate other larger questions. When they were developing these tools their work could not be published in flashy journals of science, and were generally thought to be unimaginative and not very fundable. Apparently, the US National Institutes of Health (NIH) had refused to support Capecchi’s work in 1981 and only in 1984 or 1985 could he obtain support from NIH for these studies.
Lesson: If you have a really good idea for a technology, it might be unfundable and it might not generate enough excitement immediately. The really important factor to consider, apart from the feasibility, is if you are successful will you make an incremental impact or a contribution that just was not possible before. If the latter, then beg or borrow, but do it. If the former, you’d better put on your thinking cap again. This time, first look for a big question in biology that you want solved, and then figure out a technique for solving it. Don’t put the cart (the technique) before the horse (biology or medical question).