Analysis of fiber-type differences in reporter gene expression of β-gal transgenic muscle.

Analysis of fiber-type differences in reporter gene expression of β-gal transgenic muscle.
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β-gal 转基因肌肉报告基因表达纤维类型差异的分析。

DOI:
10.1007/978-1-61779-343-1_26
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发表时间:
2012
期刊:
Methods in molecular biology (Clifton, N.J.)
影响因子:
--
通讯作者:
Hauschka,StephenD
Hauschka,StephenD
中科院分区:
--
文献类型:
--
作者:
Tai,PhillipWL;Smith,CatherineL;Angello,JohnC;Hauschka,StephenD

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β-半乳糖苷酶 (β-gal) 是研究各种生物机制(例如基因表达、细胞迁移、干细胞向不同细胞类型的转化和基因沉默)最常用的标记物之一。其中许多研究需要对安装在载玻片上并通过显微镜观察的组织横截面中的相对 β-gal 水平进行组织化学检测。这对于分析骨骼肌组织中的启动子活性特别有用,因为不同解剖肌肉和肌纤维类型之间的 β-gal 水平可能存在显着差异。启动子活性的差异可能是由于肌纤维的发育历史、神经支配、对正常或实验生理信号的反应及其疾病状态造成的。因此,识别肌肉横截面内的各个纤维类型并将其与转基因表达信号相关联非常重要。在这里,我们详细描述了如何处理和分析肌肉组织以确定冷冻切片中的纤维类型组成和 β-gal 转基因表达。
β-galactosidase (β-gal) is among the most frequently used markers for studying a wide variety of biological mechanisms, e.g., gene expression, cell migration, stem cell conversion to different cell types, and gene silencing. Many of these studies require the histochemical detection of relative β-gal levels in tissue cross-sections mounted onto glass slides and visualized by microscopy. This is particularly useful for the analysis of promoter activity in skeletal muscle tissue since the β-gal levels can vary dramatically between different anatomical muscles and myofiber types. The differences in promoter activity can be due to a myofiber’s developmental history, innervation, response to normal or experimental physiological signals, and its disease state. It is thus important to identify the individual fiber types within muscle cross-sections and to correlate these with transgene expression signals. Here, we provide a detailed description of how to process and analyze muscle tissues to determine the fiber-type composition and β-gal transgene expression within cryosections.
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