Department of Molecular Biology, Central Institute of Mental Health, Medical Faculty Mannheim/Heidelberg University, Mannheim, Germany.
PLoS One. 2012;7(5):e38193. doi: 10.1371/journal.pone.0038193. Epub 2012 May 31.
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 Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system.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.
5-羟色胺能（5-HT）神经元系统在发育和成年期具有重要且多样化的生理功能。其在发育过程中的失调或成年后的失调与许多神经精神疾病有关。为了研究 5-羟色胺易感性基因对病理性表型的贡献，设计的转基因动物模型理想情况下应允许在生命的任何所需时间选择性地在 5-羟色胺神经元中研究候选基因过表达或基因敲除。为此，条件表达系统（如 tet 系统）是首选。在这里，我们生成了一种反式激活剂（tTA）小鼠品系（TPH2-tTA），该品系允许在 5-羟色胺神经元中进行四环素（Ptet）控制的转基因表达的时空控制以及基因缺失。tTA cDNA 通过同源重组插入到含有小鼠 Tph2 片段（177kb）的 196kb PAC 中，大肠杆菌。为了功能性分析 Ptet 控制的转基因表达，TPH2-tTA 小鼠与 Ptet 调节的 lacZ 报告基因系（Ptet-nLacZ）杂交。在成年双转基因 TPH2-tTA/Ptet-nLacZ 小鼠中，TPH2-tTA 启动子线 L62-20 显示出强烈的 5-羟色胺β-半乳糖苷酶表达，该表达可以用强力霉素（Dox）完全抑制。此外，当从系统中去除或添加 Dox 时，可以可逆地激活或失活 Ptet 调节的基因表达。为了功能性分析 Ptet 控制的 Cre 介导的基因缺失，TPH2-tTA 小鼠（L62-20）与双转基因 Ptet-Cre/R26R 报告小鼠杂交，生成 TPH2-tTA/Ptet-Cre/R26R 小鼠。没有 Dox，5-HT 特异性重组于 E12.5 开始。用永久性 Dox 给药，Ptet 控制的 Cre 介导的重组不存在。出生后或成年后去除 Dox 分别诱导中缝核所有 5-羟色胺神经元中的有效重组。在肠神经系统中，无法检测到重组。我们生成了一种转基因小鼠 tTA 系（TPH2-tTA），该系允许在整个生命过程中在 5-羟色胺神经元中诱导性和可逆性地表达转基因，并诱导性 Cre 介导的基因缺失。这将允许在发育和成年期精确描绘 5-羟色胺基因的功能。
