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 September trackback spam report | Back to blogs for industry | Solid choice for Chemistry Nobel | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The 2007prize for Physiology and Medicine goes to Mario Cappechi, Oliver Smithies, and Martin Evans for their work on gene targeting in mice. The NPR explanation this morning was pretty lame. The prize is for work that allows construction of "knockout" and "knockin" mice by homologous recombination between introduced recombinant DNA molecules and chromosomal loci in embryonic stem cells, followed by generation of fertile mice by using the transgenic stem cells to generate baby mice, some of which will pass on the engineered gene to their offspring. Cappechi and Smithies both worked on the recombination part, while Evans gets credit for the work on ES cells. The problem with getting genes into mammalian cells wasn't the absence of recombination; it was the really high background of nonhomologous end-joining that led to the introduced DNA to show up as large arrays of DNA integrated randomly all over the genome. A small fraction of cells transfected with the desired DNA recombined it into the right place without simultaneously picking up these undesired DNAs in the wrong places (ectopic locations). Cappechi and Smithies each came up with a way to select for the desired cells by killing off the ones that had the wrong stuff in the wrong place. The key was to attach a conditionally toxic gene to the desired engineered gene, such that the crossovers from recombination would separate the toxic gene from the desired one. Both Capecchi and Smithies independently used the same pairs of genes: Hypoxanthine Phosphoribosyl Transferase (HPRT) for the gene they wanted to knock out and Thymidine Kinase for the toxic gene. Picking these was not just based on the human disease connection for HPRT. These genes were established as markers that could be selected for or against in mammalian tissue culture based on the knowledge of their roles in nucleoside scavanging. Neither is essential unless the de novo synthesis of purines (for hprt) or thymidine (for tk) is blocked. Both become toxic when cells are presented with toxic analogs of the appropriate nucleosides. This is the basis of the HAT (hypoxanthine aminopterin thymidine) selection for hprt+ tk+ cells developed by Smithies' Wisconsin colleague Waclaw Szybalski years before. HAT selection was important for another Wisconsin colleague, Bob DeMars, in his work in making somatic cell hybrids. I had the privilege of taking a course in eukaryotic molecular genetics from Smithies in the mid 1970s. The NYT mention that he's a private pilot understates things. He and a friend set some kind of record in a transatlantic flight, IIRC. He's an amazing and delightful scientist. Congratulations Oliver! I've met Mario Cappechi when he came to give a seminar here, but I've not met Martin Evans. It should be pointed out that the important breakthroughs in gene targeting that let to the prizewinning work were breakthroughs that were needed for knockouts in mammals. Homologous gene replacement with recombinant DNA was already working well in yeast, where the nonhomologous pathway is not significant. Ironically, a different set of technical limitations made it harder to do gene replacement in E. coli until relatively recently. Recombinant DNA molecules tend to be too small to escape E. coli systems that degrade the DNA, probably as a form of antiviral defense. Mutations in the recBCD pathway eliminated the degradation, but weren't so good at the recombination. It turns out that expressing phage recombination proteins, especially beta from phage λ, promotes efficient recombination with small linear DNA molecules.
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