A novel conditional knockout strategy applied to serotonin receptors.
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.
Stark
K L
KL
Center for Neurobiology and Behavior, Columbia University, P.I. Annex, Room 729A, 722 West 168th Street, New York, NY 10032, USA.
Gross
C
C
Richardson-Jones
J
J
Zhuang
X
X
Hen
R
R
D000818:Animals; D004318:Doxycycline / Q000494:pharmacology; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D006207:Half-Life; D009355:Neomycin / Q000494:pharmacology; D011985:Receptors, Serotonin / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*
D011985:Receptors, Serotonin; D009355:Neomycin; D004318:Doxycycline