Tetracycline Inducible Gene Manipulation in Serotonergic Neurons

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 Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system.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

Functional characterization of Ptet-controlled, Cre-mediated recombination in TPH2-tTA/Ptet-Cre/R26R mice.

<p>TPH2-tTA mice were mated with Ptet-Cre/R26R mice to generate triple-transgenic TPH2-tTA/Ptet-Cre/R26R mice. In Ptet-Cre mice, a bidirectional promoter Ptetbi allows co-regulated Ptet-controlled transcription of Cre and luciferase cDNA. Luciferase activity can be used to indirectly quantify tissue-specific Cre expression but was not needed to assess Cre-mediated recombination. In the presence of Dox (+Dox), Ptet-controlled Cre expression does not occur. In the absence of Dox (−Dox), tTA activates Ptet-controlled Cre expression. Cre then deletes a loxP-flanked stop cassette in the Rosa26 locus, thereby initiating βgal expression which indicates successful Cre-mediated recombination.</p

Non radioactive <b><i>in-situ</i></b><b> hybridization of TPH2-tTA founder lines.</b>

<p>A tTA-antisense probe was used to detect tTA-mRNA in transgenic TPH2-tTA mice. Each TPH2-tTA line was screened by non-radioactive ISH. (A-C) Exemplary tTA <i>in situ</i> hybridization of founder line 62-20 showed abundant tTA-mRNA in brain stem and midbrain areas where raphe nuclei with serotonergic neurons are located. CR, caudal raphe nuclei; DR, dorsal raphe nuclei; MR, median raphe nuclei.</p

Efficiency of Ptet-controlled βgal expression in TPH2-tTA/Ptet-nLacZ offspring of eight independent TPH2-tTA founder lines.

<p>TPH2-tTA/Ptet-nLacZ nice which had received Dox throughout development until P60 were sacrificed at P90 after 30 days of Ptet-controlled gene activation without Dox and analyzed with dual-label IHC. Efficiencies for caudal (CR), median (MR) and dorsal (DR) raphe nuclei were separately and jointly (total) calculated. Confidence-bounds (CI) were calculated using the Clopper-Pearson method based on significance level 95.0%.</p

Functional characterization of Ptet-controlled βgal expression in TPH2-tTA/Ptet-nLacZ mice.

<p>TPH2-tTA mice were mated with Ptet-nLacZ mice to generate double-transgenic TPH2-tTA/Ptet-nLacZ mice. In Ptet-nLacZ reporter mice, a bidirectional promoter Ptetbi which contains seven tet operator sequences flanked by two minimal promoters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038193#pone.0038193-Baron1" target="_blank">[77]</a> allows bidirectional Ptet-controlled transcription of nuclear localized lacZ and luciferase cDNA. Only βgal reporter expression was analyzed in TPH2-tTA/Ptet-nLacZ mice. In the presence of Dox (+Dox), βgal expression is not initiated since tTA binding to Ptet is prevented by Dox. In the absence of Dox (-Dox), tTA binds to Ptet which activates lacZ reporter gene transcription.</p