A Novel Conditional Knockout Strategy Applied to Serotonin Receptors

Abstract

Here we demonstrate the feasibility of a doubly regulatable transgenic mouse design that allows for gene manipulation by both Cre-recombinase and the tetracycline inducible system. Using a knock-in strategy to insert both elements of the tetracycline inducible system and a neomycin (neo) cassette flanked by loxP sequences (floxed) into the wild-type locus, we generated mice that express the 5-HT_1B receptor in a conditional manner. In the presence of a floxed neo-cassette, receptor expression was silenced. Removal of this cassette by Cre-mediated recombination led to 5-HT_1B receptor expression, which was highly regulatable when doxycycline, a derivative of tetracycline, was administered to the mice. This system allowed for a determination of an in vivo time course of receptor half-life and recovery. Physiological studies also demonstrated that rescued 5-HT_1B receptors were functional, and that this functionality was reversible upon treatment with doxycycline. Crossing mice where the 5-HT_1B, or the 5-HT_1A, receptors were silenced by the neo-cassette, with mice expressing either Cre-recombinase or the tetracycline transactivator (tTA) under the control of tissue-specific promoters, led to tissue-specific re-expression of these receptors. Our studies thus demonstrate the potential of this strategy for achieving both a classic knockout, as well as subsequent tissue-specific and/or inducible knockouts.