Analysis of dopamine transporter gene expression pattern - generation of DAT-iCre transgenic mice
Analysis of dopamine transporter gene expression pattern - generation of DAT-iCre transgenic mice
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DOI：
10.1111/j.1742-4658.2007.05886.x
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发表时间：
2007-07-01
期刊：
影响因子：
5.4
通讯作者：
Tronche, Francois
中科院分区：
文献类型：
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作者：
Turiault, Marc;Parnaudeau, Sebastien;Tronche, Francois
多巴胺转运蛋白是多巴胺能突触的重要组成部分。它位于突触前神经元，调节细胞外多巴胺水平。我们构建了在多巴胺转运蛋白基因调控元件控制下表达 Cre 重组酶的转基因小鼠系，用于研究多巴胺能神经元中的基因功能。密码子改良的 Cre 重组酶 (iCre) 基因被插入细菌人工染色体上的多巴胺转运蛋白基因中。免疫组织化学显示，细菌人工染色体-多巴胺转运蛋白-iCre转基因的表达模式与内源多巴胺转运蛋白基因的表达模式相似。在携带细菌人工染色体-多巴胺转运蛋白-iCre转基因和在Cre介导的重组后表达β-半乳糖苷酶基因的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）和多巴胺转运蛋白（免疫荧光）在腹侧中脑中具有相同的分布。我们利用该动物模型详细研究了下丘脑核中多巴胺转运蛋白基因表达的分布。在 A12 至 A15 中，酪氨酸羟化酶（多巴胺合成所需的酶）的表达谱比 β-半乳糖苷酶的表达谱更广泛。因此，只有一小部分合成多巴胺的神经元表达多巴胺转运蛋白基因。细菌人工染色体-多巴胺转运蛋白-iCre 转基因系是一种独特的工具，可在这些小鼠与产生荧光蛋白的转基因 Cre 报告系杂交后，将 Cre/loxP 介导的 DNA 重组靶向多巴胺神经元，以研究基因功能或标记活细胞。
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.