| Measurement of mRNA Decay in Mouse Embryonic Fibroblasts
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Author:
Date:
2016-07-05
[Abstract] mRNA stability control is a critical step in the post-transcriptional regulation of gene expression. Actinomycin D, an antibiotic initially used as an anti-cancer drug, has turned out to be a convenient tool for studying the turnover rates of transcripts in cells, due to its inhibition of mRNA synthesis. Here, we describe a protocol for the measurement of mRNA decay after adding actinomycin D into the medium of stable fibroblast cell lines derived from wild-type and tristetraprolin (TTP)-deficient mouse embryonic fibroblast (MEF) cultures, as well as a protocol for determining the relative ...
[摘要] mRNA稳定性控制是基因表达的转录后调控中的关键步骤。放线菌素D，一种最初用作抗癌药物的抗生素，由于其对mRNA合成的抑制，已经证明是用于研究细胞中转录物的转换率的方便的工具。在这里，我们描述了添加放线菌素D后的稳定成纤维细胞细胞系，从野生型和三四氯丙胺（TTP）缺陷小鼠胚胎成纤维细胞（MEF）文化，以及一个协议使用半定量实时RT-PCR确定相对转录本丰度。 Northern印迹或NanoString n-Counter是测量mRNA丰度的备选方法，在前一种情况下使用phosphorimager对其进行定量。该方案适合于研究源自转基因小鼠及其各自对照的原代培养细胞和稳定细胞系，并且提供在具有和不具有目标基因的其他相同细胞中的mRNA衰减率的直接比较。
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| Mouse Embryonic Fibroblast Cell Culture and Stimulation
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Author:
Date:
2016-07-05
[Abstract] Culture of mouse embryonic fibroblast (MEF) cells represents a powerful system to test gene function due to their easy accessibility, rapid growth rates, and the possibility of a large number of experiments. Fibroblasts are a group of heterogeneous resident cells of mesenchymal origin that have various locations, diverse appearances and distinctive activities. Because of their ubiquitous distribution as tissue cells, these cells are poised to respond to factors released by newly activated innate immune cells, thus becoming a useful tool to study inflammation and immunity. Here, we describe ...
[摘要] 小鼠胚胎成纤维细胞（MEF）细胞的培养代表了测试基因功能的强大系统，因为它们容易获得，快速生长速率和大量实验的可能性。成纤维细胞是一组具有不同位置，不同外观和独特活动的间充质来源的异质驻留细胞。由于它们作为组织细胞的普遍分布，这些细胞准备响应由新激活的先天免疫细胞释放的因子，因此成为研究炎症和免疫的有用工具。在这里，我们描述了小鼠胚胎成纤维细胞分离，原代培养和刺激的程序。具体来说，我们已经优化了刺激前血清饥饿的步骤。这个步骤是必要的，以维持这些细胞在它们暴露于促炎刺激为最佳反应之前的静止状态。如在我们以前的研究中所示，这些小鼠成纤维细胞在通过常规Northern印迹技术容易检测的水平下不表达Tnf ， Csf2 或 Lai WS等人，2006）。
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