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| Modern Genetic Analysis Chapter 11. Applications of Recombinant DNA Technology Recombinant DNA Technology in Eukaryotes Figure 11-19. Producing cells that contain a mutation in one specific gene, known as a targeted mutation or a gene knockout. (a) Copies of a cloned gene are altered in vitro to produce the targeting vector. The gene shown here has been inactivated by insertion of the neor gene (green) into a protein coding region (blue). The neor gene will serve later as a marker to indicate that the vector DNA took up residence in a chromosome. The vector has also been engineered to carry a second marker at one end: the herpes tk gene (red). These markers are standard, but others could be used instead. (b) When a vector, with its dual markers, is complete, it is introduced into cells isolated from a mouse embryo. (c) When homologous recombination occurs (top), those regions on the vector (together with any DNA in between) take the place of the original gene, excluding the marker at the tip (red). In many cells, though, the full vector (complete with the extra marker) fits itself randomly into a chromosome (middle) or does not become integrated at all (bottom). (d) To isolate cells carrying a targeted mutation, all the cells are put into a medium containing selected drugs, here a neomycin analog (G418) and ganciclovir. G418 is lethal to cells unless they carry a functional neor gene, and so it eliminates cells in which no integration of vector DNA has occurred (yellow). Meanwhile, ganciclovir kills any cells that harbor the tk gene, thereby eliminating cells bearing a randomly integrated vector (red). Consequently, virtually the only cells that survive and proliferate are those harboring the targeted insertion (green). (Redrawn from M. R. Capecchi, "Targeted Gene Replacement." Copyright © 1994 by Scientific American, Inc. All rights reserved.)

© 1999 by W. H. Freeman and Company. All rights reserved.