In 1977, Capecchi identified a long-term challenge for himself that ultimately assured his renown. He had read a paper by two Columbia University researchers, HHMI investigator Richard Axel and Michael Wigler, who made a solution of DNA with calcium phosphate; when they put it on top of a cell culture, the cells took up the DNA. Most of the time, cells' digestive enzymes destroyed it, but in about one cell in a million the DNA made it into the nucleus able to function.

Capecchi figured that a rate-limiting step in that experiment was technological—the mechanical process of getting the DNA into the nucleus—and that success would simply be a matter of innovation. “Technology itself is what makes things really jump,” he says. “All of a sudden you open up new ways of measuring things, new ways of seeing things. It's those jumps that make the significant breakthroughs in science.”

At the time, a colleague in the lab next door was doing electrophysiology with the aid of a setup that Capecchi says looked like it could be fashioned into an extremely fine hypodermic needle. He adapted the setup to create such a needle, attached DNA containing a selectable gene to a tiny fragment of viral DNA—an enhancer, though no one yet knew what it was—and then used the needle to shoot this complex into the cell nucleus. “That worked enormously efficiently,” he recalls. “About one in three cells actually picked up the DNA in functional form, so it was about a million-fold improvement in transfer of functional DNA.”

He and his students then teased out the way cells incorporated the DNA into their nuclei and found a surprise: sometimes the cells used “homologous recombination”—a physical rearrangement of genetic material between two strands of DNA—to stitch together multiple copies of the same DNA stretches one after the other. This observation proved that mammalian cells had the machinery to enable homologous recombination between copies of injected DNA molecules.

It was a small leap in imagination to envision that the same machinery could be made to bring about an exchange between a chosen piece of DNA and the similar sequence resident in the genome of the mammalian cell. In 1980, Capecchi submitted a grant proposal to the NIH for three projects, one of which would use the newly observed homologous recombination machinery to create such gene-targeting events.

“They essentially said 'No good, drop it, not likely to succeed' and also gave me one of the worst scores I'd ever received,” says Capecchi. He got the NIH funds, but, “the message was very clear: shelve gene targeting and put all your efforts into these other two projects. So I put all of our efforts into gene targeting!”

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