Conditional Knockout Mice

In the last decade, gene targeting in embryonic stem (ES) cells has been extensively used as a powerful tool to study gene function in the mouse, as a mammalian model organism. As initially developed, the technique allows the disruption of a target gene in the murine germline by the insertion of a selectable marker (). The vast majority of the more than 1000 knockout mice in existence have been created following this design. Many of these strains have given valuable information on the biologic function of the genes studied (). Since these “conventional” knockout mice are usually homozygous for a null allele in the germline, they provide an appropriate model for inherited diseases, leading to embryonic or early postnatal lethality in about 30% of cases. Apart from this application, germline knockout mice do not necessarily represent the best technical approach for studying other aspects of gene function in vivo, in particular in adult mice. A refined knockout strategy termed conditional gene targeting has been developed () that permits the inactivation of the target gene to be restricted to a certain organ and/or developmental stage (). Figure 1 depicts the principal difference between the two strategies, comparing a germline knockout mouse with two types of conditional mutants in which the target becomes inactivated either early on in a particular organ without temporal control or upon induction at a chosen time point. The inactivation of the target gene in a conditional mutant is achieved by the expression of a site-specific DNA recombinase (Cre or FLP) in mice in conjunction with the introduction of two recombinase recognition sequences [loxP or FLP recognition target (FRT)] into noncoding regions of the target gene (Fig. 2). These sites are usually placed in the same orientation into introns such that recombination results in gene inactivation through the deletion of the loxP- or FRT-flanked exon(s). Although the generation of completely ES cell-derived mice has been recently significantly improved (), the current standard method to derive a conditional mutant requires the generation of two mouse strains: one mouse strain harboring a loxP- or FRT-flanked gene segment by gene targeting in ES cells and a second transgenic strain expressing Cre or FLP either constitutively, or upon induction, in one or several organs. The conditional mutant is generated by crossing these two strains such that the inactivation of the target gene will be restricted in a spatial and temporal manner, following the pattern of Cre or FLP expression in the transgenic strain (Fig. 2, right side). As the homozygous loxP- or FRT-flanked allele must be combined with a heterozygous recombinase transgene, it often requires additional generation time (3 months) to obtain reasonable numbers of conditional mutants, compared with germline KO mice. The loxP-containing strain can be also converted into a null allele mutant by a single cross to a deleter strain expressing Cre in germ cells or the early embryo (Fig. 2, left side). There are no general rules to decide whether the germline or the conditional mutation is more appropriate for a particular experiment since this depends on the biologic question. In fact, both types of mutants are usually generated at the same time to investigate gene function both during embryonic development and in the adult animal. However, following the conditional mutagenesis scheme described in Subheading 1.4., germline and conditional mutants can be generated for a particular gene using a single targeted ES cell clone. Thus, we would recommend the conditional gene targeting scheme for all knockout projects, as this offers a greater flexibility compared with the conventional approach without additional efforts once the necessary reagents are assembled. Open image in new window Fig. 1. Gene-targeting strategies. Upper row: the knockout (KO) is transmitted through the germline, resulting in a null allele mutant strain (conventional KO); middle row: the KO is introduced in somatic cells and restricted to a specific tissue (conditional, cell type-specific KO); bottom row: the KO is introduced upon induction (conditional, inducible KO). Tissues in which the target gene is inactivated are shown in black. Open image in new window Fig. 2. Generation of a conditional mutant. Upper mouse: a strain harboring a loxP-flanked (triangles) gene segment (square). To derive a conditional mutant, the strain is crossed to mice expressing Cre recombinase constitutively or upon induction in specific cell types or organs (right side). Gene modification occurs by Cre-mediated recombination according to the expression pattern of the recombinase transgene. The loxP-containing strain can be converted into a null allele mutant by a single cross to a deleter strain expressing Cre in germ cells or the early embryo (left side).