Abstract
The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood.
Highlights
Serotonin (5-HT) is the most mysterious of the main neuromodulators [1]
The CreERT/loxP-system is used for inducible gene knockouts it allows for inducible gene expression [11]
The modified genomic insert was introduced into the mouse germline of BDF1-mice (Charles River) by oocyte injections. 17 transgenic tryptophan hydroxylase 2 (TPH2)-tTA founders were identified by PCR-genotyping of tail
Summary
Serotonin (5-HT) is the most mysterious of the main neuromodulators [1]. To delineate developmental and adult 5-HT specific gene effects on a wide spectrum of functions such as impulsivity/ behavioural disinhibition, affective control, decision making or reinforcement learning, the ability for temporal and spatial control of gene manipulation in transgenic mouse models is a prerequisite. This is important when timely manipulations of the 5HT system are to be combined with specific environmental interventions such as maternal deprivation or housing isolation during development or adulthood. The tet-system is widely used for inducible and reversible expression of candidate genes albeit temporal control of tissue-specific gene deletion is possible [12]
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