| Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells
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Author:
Date:
2016-02-20
[Abstract] In this protocol we describe our modifications to a method to isolate, culture and maintain mouse intestinal stem cells as crypt-villus forming organoids. These cells, isolated either from the small or large intestine, maintain self-renewal and multilineage differentiation potential over time. This provides investigators a tool to culture wild type or transformed intestinal epithelium, and a robust assay for stem cell tissue homeostasis in vitro.
[摘要] 在这个协议，我们描述我们修改分离，培养和维持鼠肠干细胞作为隐窝 - 绒毛形成类器官的方法。 从小肠或大肠分离的这些细胞随时间保持自我更新和多向分化潜能。 这为研究人员提供了培养野生型或转化的肠上皮的工具，以及用于体外干细胞组织稳态的稳健试验。
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| Measurement of 2-methylthio Modifications in Mitochondrial Transfer RNAs by Reverse-transcription Quantitative PCR
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Author:
Date:
2016-01-05
[Abstract] 2-Methylthio-N6-isopentenyladenosine (ms2i6A) is an evolutionally conserved posttranscriptional modification found at position 37 of four mammalian mitochondrial tRNAs, mt-tRNASer(UCN), mt-tRNATrp, mt-tRNAPhe and mt-tRNATyr. The ms2 modification in ms2i6A strengthens codon-anticodon interaction and contributes to accurate and efficient decoding. Deficiency of ms2 modifications impairs mitochondrial protein synthesis, which ultimately leads to the development of myopathy in mice and patients having mitochondrial diseases. Therefore, the level of ms2 could be utilized as an indicator that reflects the status of mitochondrial protein synthesis. Here, we ...
[摘要] 2-甲硫基 - N 6 - 叔丁基腺苷（ms）是一种进化保守的转录后修饰发现 在四个哺乳动物线粒体tRNA，mt-tRNA Ser（UCN），mt-tRNA Trp ，mt-tRNA < trna=""> Tyr 。 ms 2 6中的ms 2修饰加强了密码子 - 反密码子相互作用，并有助于准确和有效的解码。 缺乏修复损害线粒体蛋白质合成，其最终导致小鼠和患有线粒体疾病的患者中的肌病的发展。 因此，ms 2的水平可以用作反映线粒体蛋白质合成状态的指标。 在这里，我们描述了一种简单和快速的定量PCR为基础的方法来测量总RNA样本中的ms 2水平。
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| Purification and TEM of Afp and Its Variants
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Author:
Date:
2014-05-20
[Abstract] The Serratia entomophila antifeeding prophage (Afp), forms a phage-tail-like particle (tailocin) that causes cessation of feeding activity of the New Zealand grass grub, Costelytra zealandica. Here, we describe the more detailed purification protocol for Afp particles and its variants which is based on the procedure described in our original publication (Rybakova et al., 2013). The purification procedure includes inducing Escherichia coli (E. coli) cells harbouring afp genes under arabinose-inducible promoter for 24 h. The cells are harvested and sonicated on ice followed by DNAse treatment and centrifugation. The supernatant is then filter sterilised and applied to the size exclusion chromotography (SEC) column. The fractions ...
[摘要] 噬菌体噬菌体抗原噬菌体（Afp）形成噬菌体尾样颗粒（太古霉素），其导致新西兰草禾本科（Costelytra zealandica）的饲养活性的停止。在这里，我们描述了Afp颗粒及其变体的更详细的纯化方案，其基于我们的原始出版物中描述的程序（Rybakova等人，2013）。纯化程序包括在阿拉伯糖诱导型启动子下诱导含有emp 基因的大肠杆菌（大肠杆菌）细胞24小时。收获细胞，在冰上超声处理，然后进行DNA酶处理和离心。然后将上清液过滤灭菌，并应用于尺寸排阻色谱（SEC）柱。合并含有Afp或其变体的级分并超速离心。除去上清液，将透明沉淀物重悬浮于残余缓冲液中。该程序产生约70％纯的Afp颗粒或其变体。使用透射电子显微镜（TEM）对Afp颗粒进行负染色并可视化。
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