ABSTRACT
Gene targeting can be used in embryonic stem cells to produce strains of genetically modified mice. Studies employing targeted gene inactivation are making a major contribution to the understanding of mammalian gene function and to the modelling of human inherited genetic deficiency diseases in the mouse. We have developed a procedure called double-replacement gene targeting, which uses hypoxanthine phosphoribosyltransferase (HPRT) selectable markers in HPRT-deficient embryonic stem cells for the production of mice with subtle gene alterations, rather than conventional gene inactivations (knockouts). In the first targeting step, the target locus is inactivated by deleting part of the gene and replacing it with an HPRT selectable marker. In the second step, the HPRT marker is itself replaced by a modified version of the original gene segment, which contains the desired subtle gene alteration. From the initial targeting event, this procedure can be used to create a series of mouse strains with different subtle gene alterations.
