Turiault Marc, Parnaudeau Sébastien, Milet Aude, Parlato Rosanna, Rouzeau Jean-Denis, Lazar Monique, Tronche François
CNRS UMR7148, Molecular Genetics, Neurophysiology and Behavior, Collège de France, Institut de Biologie, Paris, France Molecular Biology of the Cell I, German Cancer Research Center, Heidelberg, Germany.
FEBS J. 2007 Jul;274(14):3568-3577. doi: 10.1111/j.1742-4658.2007.05886.x. Epub 2007 Jun 12.
The dopamine transporter is an essential component of the dopaminergic synapse. It is located in the presynaptic neurons and regulates extracellular dopamine levels. We generated a transgenic mouse line expressing the Cre recombinase under the control of the regulatory elements of the dopamine transporter gene, for investigations of gene function in dopaminergic neurons. The codon-improved Cre recombinase (iCre) gene was inserted into the dopamine transporter gene on a bacterial artificial chromosome. The pattern of expression of the bacterial artificial chromosome-dopamine transporter-iCre transgene was similar to that of the endogenous dopamine transporter gene, as shown by immunohistochemistry. Recombinase activity was further studied in mice carrying both the bacterial artificial chromosome-dopamine transporter-iCre transgene and a construct expressing the beta-galactosidase gene after Cre-mediated recombination. In situ studies showed that beta-galactosidase (5-bromo-4-chloroindol-3-yl beta-D-galactoside staining) and the dopamine transporter (immunofluorescence) had identical distributions in the ventral midbrain. We used this animal model to study the distribution of dopamine transporter gene expression in hypothalamic nuclei in detail. The expression profile of tyrosine hydroxylase (an enzyme required for dopamine synthesis) was broader than that of beta-galactosidase in A12 to A15. Thus, only a fraction of neurons synthesizing dopamine expressed the dopamine transporter gene. The bacterial artificial chromosome-dopamine transporter-iCre transgenic line is a unique tool for targeting Cre/loxP-mediated DNA recombination to dopamine neurons for studies of gene function or for labeling living cells, following the crossing of these mice with transgenic Cre reporter lines producing fluorescent proteins.
多巴胺转运体是多巴胺能突触的重要组成部分。它位于突触前神经元中，调节细胞外多巴胺水平。我们构建了一种转基因小鼠品系，其在多巴胺转运体基因调控元件的控制下表达Cre重组酶，用于研究多巴胺能神经元中的基因功能。将密码子优化的Cre重组酶（iCre）基因插入细菌人工染色体上的多巴胺转运体基因中。免疫组织化学结果显示，细菌人工染色体-多巴胺转运体-iCre转基因的表达模式与内源性多巴胺转运体基因相似。在同时携带细菌人工染色体-多巴胺转运体-iCre转基因和Cre介导重组后表达β-半乳糖苷酶基因构建体的小鼠中，进一步研究了重组酶活性。原位研究表明，β-半乳糖苷酶（5-溴-4-氯吲哚-3-基β-D-半乳糖苷染色）和多巴胺转运体（免疫荧光）在腹侧中脑具有相同的分布。我们利用这个动物模型详细研究了多巴胺转运体基因在下丘脑核团中的表达分布。在A12至A15区域，酪氨酸羟化酶（多巴胺合成所需的一种酶）的表达谱比β-半乳糖苷酶更广泛。因此，只有一部分合成多巴胺的神经元表达多巴胺转运体基因。细菌人工染色体-多巴胺转运体-iCre转基因品系是一种独特的工具，可将Cre/loxP介导的DNA重组靶向多巴胺神经元，用于基因功能研究或在将这些小鼠与产生荧光蛋白的转基因Cre报告品系杂交后标记活细胞。
