Targeted gene expression in dopamine and serotonin neurons of the mouse brain
Introduction
Aberrant functioning of the ascending dopaminergic or serotonergic systems is prominent in many neuropsychiatric disorders including schizophrenia, Parkinson's disease, attention deficit hyperactivity disorder, drug addiction, depression, and anxiety. A striking feature of these systems is that they arise from just a few hundred thousand neurons concentrated in brainstem nuclei sending projections to widely distributed target areas (Cooper et al., 1996). We have taken advantage of the dopamine transporter (DAT) and serotonin transporter (SERT), known specific markers of dopaminergic and serotonergic neurons, respectively (Augood et al., 1993, Hensler et al., 1994), and used their promoters to generate cell-specific Cre recombinase expressing mice.
The Cre-loxP system has become a standard approach for performing region-specific gene inactivation in mice (Kuhn and Torres, 2002). However, this strategy has not previously been used to generate gene knockouts in specific neurotransmitter systems. Traditionally, transgenic lines are generated via pronuclear injection of the transgene that will be integrated into the genome randomly. The level and pattern of transgene expression are determined both by the promoter and by its integration site in the genome. Consequently, such transgenic mice often have transgene expression in regions or cell types not associated with the original specificity of the promoter (Bronson et al., 1996, Wallace et al., 2000). In order to direct the expression of the Cre recombinase more precisely to specific monoaminergic cell types, we used a knock-in approach to place the transgene downstream of the endogenous promoters of DAT and SERT.
Mice engineered using such an approach express Cre recombinase in dopaminergic and serotonergic neurons. Crossing these cre mice with reporter lines, demonstrated the high efficiency and specificity of this approach. Moreover, these Cre-expressing lines were used successfully to label dopamine and serotonin neurons in vivo with yellow fluorescent protein (YFP), allowing for visualization of the living neurons in brain slices. Our results represent the first example in which Cre recombinase activity is restricted to a specific monoamine neurotransmitter system and provide a general strategy that can be used to target mutations and fluorescent markers to specific neurotransmitter systems.
Section snippets
Generation of transgenic mice
DAT and SERT genomic DNA fragments that contained the 5′-region and the first two exons were excised from phage DNA isolated from a mouse 129 Sv/J genomic library. The gene targeting vectors were constructed by inserting into the 5′-UTR region of the DAT and SERT genes a cassette containing the Cre recombinase coding sequence with a nuclear localization signal and the neomycin-resistance gene, flanked by FRT sites (Fig. 1a and c). W9.5 embryonic stem (ES) cells were electroporated (Bio-Rad Gene
Cre recombinase expression in dopaminergic and serotonergic neurons in DAT-cre and SERT-cre mice, respectively
In order to direct the expression of the Cre recombinase precisely to dopaminergic and serotonergic neurons, the cre transgene was inserted downstream of the endogenous promoter of DAT (DAT-cre mice) and SERT (SERT-cre mice). Fig. 1a and c illustrate the gene-targeting constructs. Immunohistochemical staining of adult brain sections revealed Cre recombinase expression in the VAT and SNc in DAT-cre mice and in the Raphe nuclei in SERT-cre mice (Fig. 1b and d). In addition to midbrain
Discussion
The promoters of the serotonin and dopamine transporters were used to drive Cre recombinase expression in order to achieve precise neuron-specific Cre recombinase activity. The most important application of these DAT-cre and SERT-cre mice will be to target mutations specifically to dopaminergic and serotonergic neurons, respectively. For example, DAT-cre mice will allow us to perform knockout of dopamine D2 receptors specifically in dopamine neurons (i.e. D2 autoreceptor specific knockout). In
Acknowledgements
This work was support in part by The American Parkinson Disease Association and Parkinson's Disease Foundation (to X.Z.), NIMH (to R.H.), NIDA and NARSAD (to S.R.). We would like to thank Frank Costantini for kindly providing us the ROSA-26 YFP reporter mice, Joseph Gogos for providing us the nCreFNF vector, Monica Mendelsohn for blastocyst injection of ES cells, and Indira Mendez for technical assistance. Requests for the DAT-cre and SERT-cre mice should be sent to X.Z.
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Present address: Faculte de Medecine Pitie Salpetriere, 91 Bd de l’Hopital, 75634 Paris Cedex 13, France.