In Celebration of Dr. Mario R. Capecchi's Nobel Prize

Smithies for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem (ES) cells. This technology, commonly referred to as gene targeting or knockout, is based on homologous recombination between DNA sequences residing in the chromosome and newly introduced DNA to mutate any genes of interesting in mouse genome [1]. Twenty years ago, Capecchi and Smithies reported the targeted disruption of the hypoxanthine-guanine phosphoribosyl transferase gene (Hprt) [2], and the targeted correction of a defective Hprt allele [3], respectively, using the ES technology developed by Evans [4]. Since then, gene targeting has proven to be a powerful means of precisely manipulating the mammalian genome, generating at least ten thousand mutant mouse stains. Studies of these mutant mice have yielded enormously useful information in virtually all fields of biological and biomedical sciences. Indeed, gene targeting in ES cells can theoretically be used to generate mutant mice for all genes in the near future. Dr. Capecchi's groundbreaking work for gene targeting started well before 1987. In 1980, he demonstrated high efficiency of transformation by directly microinjecting plasmid DNA into cultured mammalian cells [5]. This work led to his important conclusion that mammalian somatic cells possess an efficient enzymatic machinery for mediating homologous recombination [6]. He reasoned that if this machinery could be efficient for homologous recombination between a newly introduced DNA molecule and the endogenous DNA sequence, any host gene could be mutated. To demonstrate this principle, he generated recipient cell lines carrying an artificial target, a defective neomycin resistance gene that was stably integrated into host chromosome, and was able to repair it by injecting the same gene carrying a different mutation into the nucleus of these cells [7]. The correction occurred in one cell per 1,000 injected cells, a relatively high frequency, which makes it possible to use homologous recombination to manipulate endogenous genes of the mammalian genome. These studies led to his successful targeted disruption of the first endogenous gene, Hprt, in ES cells in 1987, as mentioned earlier [2]. In this important study, Drs. Thomas and Capecchi used both replacement and insertional vectors to introduce a neomycin resistance (neo r) gene into exon 8 of the Hprt gene, showing that clones of transfected cells had lost HPRT function (becoming 6-TG resistant), and had gained Neo r activity. Therefore, the HPRT-deficient cells could be easily selected. In their paper, they concluded "It …