Conditional gene targeting in mouse pancreatic ß-Cells: analysis of ectopic Cre transgene expression in the brain.
Conditional gene targeting in mouse pancreatic ß-Cells: analysis of ectopic Cre transgene expression in the brain.
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DOI：
10.2337/db10-0624
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发表时间：
2010-12
期刊：
影响因子：
7.7
通讯作者：
Dempsey PJ
中科院分区：
文献类型：
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作者：
Wicksteed B;Brissova M;Yan W;Opland DM;Plank JL;Reinert RB;Dickson LM;Tamarina NA;Philipson LH;Shostak A;Bernal-Mizrachi E;Elghazi L;Roe MW;Labosky PA;Myers MG Jr;Gannon M;Powers AC;Dempsey PJ
条件基因靶向已广泛用于β细胞生物学中基因功能的体内分析。本研究的目的是检查用于介导β细胞或胰腺特异性重组的小鼠转基因Cre系是否也驱动Cre在脑中的表达。将由Ins 1、Ins 2和Pdx 1启动子驱动的转基因Cre系培育成R26 R报告菌株。通过胰腺和脑中的β-半乳糖苷酶或黄色荧光蛋白表达来评估Cre活性。使用Pdx 1 tm 1Cvw lacZ敲入小鼠监测内源性Pdx 1基因表达。通过免疫组织化学评估β细胞中的Cre表达以及Cre活性与脑内食欲素表达和瘦素反应神经元的共定位。所有使用Ins 2启动子驱动Cre表达的转基因Cre系在大脑中表现出广泛的Cre活性，而使用Pdx 1启动子片段的Cre系主要在下丘脑中表现出更受限制的Cre活性。免疫组织化学分析显示，从Tg（Pdx 1-cre）89.1大坝小鼠下丘脑的神经元表达食欲素和瘦素激活的神经元中的Cre活性。Tg（Ins 1-Cre/ERT）1 Lphi小鼠是唯一一个大脑中缺乏Cre活性的品系。Cre介导的基因操作使用转基因株系，表达Cre的Ins 2和Pdx 1启动子的控制下，可能会改变营养感应神经元的基因表达。因此，必须仔细解释使用这些转基因Cre系产生的数据，以评估所得表型是否仅归因于胰岛β细胞的改变。
Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell– or pancreas-specific recombination, also drive Cre expression in the brain. Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1tm1Cvw lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry. All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)89.1Dam mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)1Lphi mice were the only line that lacked Cre activity in the brain. Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells.