Refined protocols of tamoxifen injection for inducible DNA recombination in mouse astroglia
Refined protocols of tamoxifen injection for inducible DNA recombination in mouse astroglia
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DOI：
10.1038/s41598-018-24085-9
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发表时间：
2018-04-12
影响因子：
4.6
通讯作者：
Kirchhoff, Frank
中科院分区：
文献类型：
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作者：
Jahn, Hannah M.;Kasakow, Carmen V.;Kirchhoff, Frank
他莫昔芬 (TAM) 肝脏代谢物在体内诱导 floxed 等位基因 DNA 重组是研究基因功能的重要工具。在这里，我们描述了基于 GLAST-Cre(ERT2)/loxP 系统的星形胶质细胞中最佳 DNA 重组的方案。此外，我们证明基因组重组的量化可以确定不同大脑区域中细胞类型的比例。我们通过液相色谱-高分辨率串联质谱法 (LC-HR-MS/MS) 分析了小鼠大脑和血清中 TAM 及其代谢物（N-去甲基他莫昔芬、4-羟基他莫昔芬和艾多昔芬）的存在和清除情况，并通过几个 floxed 靶基因（p2ry1、p2ry1、 gria1、gabbr1 和 Rosa26-tdTomato 基因座）。通过每天注射一次，连续五天和连续三天，可以在皮层和小脑中实现最大重组。此外，量化 floxed 等位基因的丢失可以预测每个大脑区域 GLAST 阳性细胞（星形胶质细胞）的百分比。我们发现，在皮质、海马、脑干和视神经中，星形胶质细胞占细胞总数的20%~30%，而在小脑伯格曼胶质细胞中，帆状星形胶质细胞和白质星形胶质细胞仅占所有细胞的8%。
Inducible DNA recombination of floxed alleles in vivo by liver metabolites of tamoxifen (TAM) is an important tool to study gene functions. Here, we describe protocols for optimal DNA recombination in astrocytes, based on the GLAST-Cre(ERT2)/loxP system. In addition, we demonstrate that quantification of genomic recombination allows to determine the proportion of cell types in various brain regions. We analyzed the presence and clearance of TAM and its metabolites (N-desmethyl-tamoxifen, 4-hydroxytamoxifen and endoxifen) in brain and serum of mice by liquid chromatographic-high resolution-tandem mass spectrometry (LC-HR-MS/MS) and assessed optimal injection protocols by quantitative RT-PCR of several floxed target genes (p2ry1, gria1, gabbr1 and Rosa26-tdTomato locus). Maximal recombination could be achieved in cortex and cerebellum by single daily injections for five and three consecutive days, respectively. Furthermore, quantifying the loss of floxed alleles predicted the percentage of GLAST-positive cells (astroglia) per brain region. We found that astrocytes contributed 20 to 30% of the total cell number in cortex, hippocampus, brainstem and optic nerve, while in the cerebellum Bergmann glia, velate astrocytes and white matter astrocytes accounted only for 8% of all cells.