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用于诱导小鼠神经胶质细胞中 DNA 重组的他莫昔芬注射的改良方案。

Refined protocols of tamoxifen injection for inducible DNA recombination in mouse astroglia.

机构信息

Molecular Physiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), University of Saarland, 66421 Homburg, Germany.

Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PL, UK.

出版信息

Sci Rep. 2018 Apr 12;8(1):5913. doi: 10.1038/s41598-018-24085-9.

DOI:10.1038/s41598-018-24085-9
PMID:29651133
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5897555/
Abstract

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/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.

摘要

诱导 floxed 等位基因在体内的可诱导 DNA 重组通过他莫昔芬 (TAM) 的肝代谢物是研究基因功能的重要工具。在这里，我们基于 GLAST-Cre/loxP 系统描述了在星形胶质细胞中进行最佳 DNA 重组的方案。此外，我们证明了基因组重组的定量分析可以确定各种脑区中细胞类型的比例。我们通过液相色谱-高分辨率串联质谱 (LC-HR-MS/MS) 分析了 TAM 及其代谢物（N-去甲基他莫昔芬、4-羟基他莫昔芬和内消旋他莫昔芬）在小鼠脑和血清中的存在和清除情况，并通过对几个 floxed 靶基因 (p2ry1、gria1、gabbr1 和 Rosa26-tdTomato 基因座) 的定量 RT-PCR 评估了最佳注射方案。通过每天单次注射，可分别在皮层和小脑实现最大的重组，持续 5 天和 3 天。此外，定量 floxed 等位基因的丢失可以预测每个脑区的 GLAST 阳性细胞（星形胶质细胞）的百分比。我们发现星形胶质细胞在皮层、海马体、脑干和视神经中占总细胞数的 20%至 30%，而在小脑的蒲肯野细胞、软脑膜星形胶质细胞和白质星形胶质细胞仅占所有细胞的 8%。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f4/5897555/0698b053e6db/41598_2018_24085_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f4/5897555/b66be0e62e78/41598_2018_24085_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f4/5897555/1eff29cf3004/41598_2018_24085_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f4/5897555/6b239a648378/41598_2018_24085_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f4/5897555/381f100100ee/41598_2018_24085_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f4/5897555/0698b053e6db/41598_2018_24085_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f4/5897555/b66be0e62e78/41598_2018_24085_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f4/5897555/1eff29cf3004/41598_2018_24085_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f4/5897555/6b239a648378/41598_2018_24085_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f4/5897555/381f100100ee/41598_2018_24085_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78f4/5897555/0698b053e6db/41598_2018_24085_Fig5_HTML.jpg

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