Received October 31, 2002; Accepted November 15, 2002. Author to whom all correspondence and reprint requests should be addressed: Dr. Ravi P. Misra, Department of Biochemistry, Medical College of Wis- consin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail: rmisra @mcw.edu Gene Targeting in the Mouse Advances in Introduction of Transgenes into the Genome by Homologous Recombination Ravi P. Misra and Stephen A. Duncan 1 Departments of Biochemistry and 1 Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI Endocrine, vol. 19, no. 3, 229–238, December 2002 0969–711X/02/19:229–238/$20.00 © 2002 by Humana Press Inc. All rights of any nature whatsoever reserved. 229 The ability to stably introduce genes into the germline of animals provides a powerful means to address the genetic basis of physiology. Introduction of genes to generate transgenic animals has facilitated the devel- opment of complex genetic models of disease, as well as the in vivo study of gene function. However, one draw- back of traditional transgenic technologies in which genes are microinjected into early-stage embryos is that there is little control over where and in how many copies genes are introduced into the genome. The development of animal transgenic technologies, which take advan- tage of homologous recombination mechanisms and the manipulation of embryonic stem (ES) cells, allows inves- tigators to target and alter specific loci. In mouse trans- genic systems, a plethora of sophisticated gene-target- ing strategies now permit investigators to manipulate the genome in ways that essentially allow one to intro- duce virtually any desired change into the genome. Fur-thermore, when coupled with systems that allow for conditional gene expression, these gene-targeting strategies allow both temporal and tissue specific con- trol of alterations to the genome. In the present review we briefly discuss some of the more recent gene-tar- geting strategies that have been developed to address the limitations of traditional animal transgenesis. Key Words: Gene targeting; homologous recombination. Transgenic Approaches to Studying Gene Function In Vivo Techniques for introducing genes stably into the germ- line of experimental mammals provide a powerful means to investigate complex biological phenomena and to gener- ate animal models of disease. In the most common approaches, genes are introduced or altered either by genetic manipu- lation of embryonic stem (ES) cells or by microinjection of DNA into the male pronucleus of fertilized eggs. Micro- injection-based approaches are well suited to examining the physiological effects of overexpression or misexpression of gene products of interest. Such approaches also allow analysis of transcriptional regulatory elements in vivo. While a large number of studies have used a pronuclear injection approach to address gene expression and physiological function,there exist limitations on its usefulness. Notably, pronuclear injection results in an unpredictable number of transgene copies being incorporated at random locations in the recipient genome. This can result in ectopic expression of the transgene because both copy number and the site of integration can influence transgene expression. This can be a particular disadvantage in gene expression studies, which rely on independent analysis of multiple cis-acting regula- tory regions. To account for these effects, multiple inde- pendent lines of transgenic mice need to be established for each experiment. Another limitation is presented by expres- sion of transgenes that confer developmental lethal phe- notypes. In such cases no founder line can be established, and, therefore, it is impossible to repeat an analysis with a specific transgenic mouse embryo. In part, to address these limitations various strategies have been developed that typi- cally rely on targeting transgenes to a specific genomic locus by homologous recombination in pluripotent ES cells, thereby allowing precise modification of the gene of interest (1). These ES cell lines can then be used to generate transgenic animals (Fig. 1) (2). Using these approaches, it is possible to create mice of virtually any genotype. In addition, such gene-targeting methodologies can be coupled with bacteri- ally derived site-specific recombination systems that allow introduction of loss-of-function mutations in the context of both developing and intact animals. Gene targeting strate- gies that create such “knock-out” mice having a null geno- type often provide definitive experimental evidence regard- ing the functions of encoded proteins, and strategies that knockout genes “conditionally” can address developmen- tal lethal phenotypes. The principles of gene targeting were first established in experiments with Saccharomyces cerevisiae. During this time many of the basic tools that facilitated gene targeting in eukar-