A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse

Functional redundancies, compensatory mechanisms, and lethal phenotypes often prevent the full analysis of gene functions through generation of germline null mutations in the mouse1. The use of site-specific recombinases, such as Cre, which catalyzes recombination between loxP sites2, has allowed the engineering of mice harboring targeted somatic mutations, which are both temporally controlled and cell-type restricted1,3. Many Creexpressing mouse lines exist, but only a few transgenic lines are available that harbor a reporter gene whose expression is dependent on a Cre-mediated event3. Moreover, their use to monitor gene ablation at the level of individual cells is often limited, as in some tissues the reporter gene may be silenced1, be affected by position-effect variegation4, or reside in a chromatin configuration inaccessible for recombination5. Thus, one cannot validly extrapolate from the expression of a reporter transgene to an identical ablation pattern for the conditional allele of a given gene. By combining the ability of Cre recombinase to invert or excise a DNA fragment, depending on the orientation of the flanking loxP sites6, and the availability of both wild-type (WT) and mutant loxP sites7, we designed a Cre-dependent genetic switch (FLEx switch) through which the expression of a given gene is turned off, while the expression of another one is concomitantly turned on. We demonstrate the efficiency and reliability of this switch to readily detect, in the mouse, at the single cell level, Cre-mediated gene ablation. We discuss how this strategy can be used to generate genetic modifications in a conditional manner.