| In vitro Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (in vitro enChIP) Using CRISPR Ribonucleoproteins in Combination with Next-generation Sequencing (in vitro enChIP-Seq) for the Identification of Chromosomal Interactions
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Author:
Date:
2017-11-20
[Abstract] We have developed locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies consisting of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP). Locus-specific ChIP is a method to isolate a genomic region of interest from cells while it also identifies what binds to this region using mass spectrometry (for protein) or next generation sequencing (for RNA or DNA) as described in Fujita et al. (2016a). Recently, we identified genomic regions that physically interact with a locus using an updated form of enChIP, in vitro enChIP, in combination with NGS (in vitro enChIP-Seq) (Fujita et al., 2017a). Here, we describe a protocol on in vitro enChIP to isolate a target locus for identification of ...
[摘要] 我们开发了基因座特异性染色质免疫沉淀（基因座特异性芯片）技术，包括插入ChIP（iChIP）和工程DNA-结合分子介导ChIP（enChIP）。 基因座特异性ChIP是一种从细胞中分离感兴趣的基因组区域的方法，同时它还使用质谱（用于蛋白质）或下一代测序（用于RNA或DNA）鉴定什么与该区域结合，如Fujita等人 （2016a）。 最近，我们使用更新后的enChIP形式，结合NGS（ in vitro enChIP-Seq），鉴定了与基因座物理相互作用的基因组区域（Fujimita et al。，2017a）。 在这里，我们描述了一个体外试验的方法，用于分离靶基因座以鉴定与基因座物理相互作用的基因组区域。
【背景】阐明基因组功能强调的分子机制需要鉴定与感兴趣的基因组区域相互作用的分子。为此，我们开发了由插入ChIP（iChIP）和工程化DNA结合分子介导的ChIP（enChIP）组成的基因座特异性染色质免疫沉淀技术（基因座特异性ChIP）技术（Fujita等人 ...
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| Human Astrovirus Propagation, Purification and Quantification
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Author:
Date:
2014-03-20
[Abstract] Astrovirus are small, nonenveloped, single-stranded RNA viruses that cause diarrhea in a wide variety of mammals and birds. Here, we describe astrovirus propagation, purification and titration. The Caco-2 human intestinal adenocarcinoma cell line is most widely used for studying astrovirus, although other cell lines, such as 293, T84 and LLC-MK2 can be used for propagation. However, Caco2 cells are desirable for their ability to form a differentiated intestinal epithelium, mimicking the human intestine and providing a realistic model for astrovirus growth and propagation.
[摘要] 星状病毒是小的，未发育的单链RNA病毒，其在多种哺乳动物和鸟类中引起腹泻。 在这里，我们描述星状病毒繁殖，纯化和滴定。 Caco-2人肠腺癌细胞系最广泛地用于研究星形病毒，尽管其它细胞系，例如293，T84和LLC-MK2可用于繁殖。 然而，Caco2细胞是其形成分化的肠上皮的能力，模拟人的肠并且为星状病毒生长和繁殖提供现实模型所需的。
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| Mouse ESC Differentiation to Nkx2.1+ Lung and Thyroid Progenitors
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Author:
Date:
2012-11-20
[Abstract] The de novo derivation of lung progenitors from pluripotent stem cells provides the opportunity to model early lung development in vitro and allows easy access to cells for tissue engineering or basic cell biology studies. This detailed protocol allows the generation of lung and thyroid progenitors from mouse embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC) lines. When used together with a published Nkx2.1-GFP knock-in ESC line, the protocol allows tracking and purification of lung and thyroid progenitors by sorting on the GFP reporter based on the induction of the earliest known marker of lung and thyroid cell fate, Nkx2.1. After sorting, a pure population of Nkx2.1+ cells can then be replated for further expansion, differentiation, and maturation in ...
[摘要] 来自多能干细胞的肺祖细胞的新发现提供了在体外模拟早期肺发育的机会，并且容易获得用于组织工程或基础细胞生物学研究的细胞。 该详细方案允许从小鼠胚胎干细胞（ESC）或诱导多能干细胞（iPSC）系生成肺和甲状腺祖细胞。 当与公布的Nkx2.1-GFP敲入ESC系一起使用时，该方案允许通过基于对最早已知的肺和甲状腺细胞命运的标记物的诱导在GFP报告基因上进行分选来跟踪和纯化肺和甲状腺祖细胞， Nkx2.1。 在分选后，然后可以在无血清条件下培养纯化的Nkx2.1 +细胞群，以进一步扩增，分化和成熟。
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