| Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery
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Author:
Date:
2017-04-05
[Abstract] The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair mechanism. However, in presence of a DNA repair template, Homology-Directed Repair (HDR) can occur leading to precise repair of the lesion site. This last process can be exploited to enable precise knock-in changes by introducing the desired genomic alteration on the repair template. In this protocol we describe the delivery of long repair templates (> 200 nucleotides) using recombinant Adeno Associated Virus (rAAV) for CRISPR-Cas9-based knock-in of a ...
[摘要] 可编程集群定期间隔短回归度（CRISPR）相关核酸酶9（Cas9）技术通过提供在所需位置切割基因组的有效方式，彻底改变了基因组编辑（Ledford，2015）。 在哺乳动物细胞中，DNA损伤触发易发生非同源末端连接（NHEJ）DNA修复机制。 然而，在DNA修复模板的存在下，可以发生同源性定向修复（HDR），导致病变部位的精确修复。 可以利用最后的方法，通过在修复模板上引入所需的基因组改变来实现精确的敲入变化。 在本协议中，我们描述了使用重组腺相关病毒（rAAV）在人细胞系中进行基于CRISPR-Cas9的C-末端标签序列敲入的长修复模板（> 200个核苷酸）的递送。
尽管有关CRISPR-Cas9产生的敲门模型系统的大量报告，敲门砖报告仍然落后。由于许多应用，产生敲入细胞系仍然是基因组编辑的明显目标。敲入改变的引入通常依赖于修复模板DNA的存在，并且在位点特异性双链（ds）DNA断裂被引入接近改变位点的基因组中后，HDR修复机制的激活。不同的模板可以传送到修复机器，范围从含有广泛同源区域和可选选择盒的经典线性化载体到约200个核苷酸的单链（ss）DNA寡核苷酸（Chen等人， ...
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| Determination of Rate of [3H-methyl]-choline Incorporation into Cellular Lipids and Non-lipid Metabolites
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Author:
Date:
2016-11-20
[Abstract] The choline-containing phospholipid, phosphatidylcholine (PtdCho) is the most common mammalian phospholipid found in cell membrane (Ide et al., 2013). It is also a component of intracellular signalling pathways (Cui and Houweling, 2002). Herein is described a measure of the rate of accumulation of choline by lipid soluble PtdCho and lyso-Ptdcho which can further be discriminated by chromatographic analysis (Smith and Phyu, 2016). Determination of the accumulation of [3H-methyl]-choline into water-soluble components is also described. The procedure could be used to measure the effect of drugs and other factors on choline incorporation into phospholipids. After exposure of cells to test conditions (e.g., drugs) adherent cells in tissue culture flasks are ...
[摘要] 含胆碱的磷脂，磷脂酰胆碱（PtdCho）是在细胞膜中发现的最常见的哺乳动物磷脂（Ide等人，2013）。它也是细胞内信号传导途径的组成部分（Cui和Houweling，2002）。本文描述了脂质可溶性PtdCho和溶血磷脂酰胆碱积累胆碱的速率的量度，其可通过色谱分析进一步区分（Smith和Phyu，2016）。还描述了[3 H] - 甲基] - 胆碱在水溶性组分中的积累的测定。该程序可用于测量药物和其它因素对胆碱掺入磷脂的影响。在将细胞暴露于测试条件（例如，药物）后，将组织培养瓶中的贴壁细胞与放射性标记的[3 H] - 甲基] - 胆碱在培养基中温育15分钟（脉冲）。然后快速除去[3 H] - 甲基] - 胆碱，并在非放射性培养基（Chase）存在下继续孵育。然后通过细胞分级和测量脂质和非脂质细胞组分中的放射性来测定[3 H] - 甲基的细胞分布。
[背景] 磷脂代谢在细胞膜形成中是必需的（Ide等人，2013）和细胞信号传导（Cui和Houweling， 2002）。包含胆碱的代谢物的形成和胆碱在脂质中的积累是细胞增殖过程中的关键过程。磷脂代谢紊乱与癌症和其他疾病有关（Gibellini和Smith，2010）。这些过程的测量对于理解医学成像模态至关重要，所述医学成像模式使用[11 C] - ...
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| Isolation of Intestinal Mesenchymal Cells from Adult Mice
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Author:
Date:
2016-09-20
[Abstract] During the last 20 years intestinal mesenchymal cells (IMCs) have emerged as an important cell type that plays a central role in intestinal development and homeostasis, by providing both structural support and growth regulatory elements. IMCs also actively participate in wound healing responses, thus regulating pathologic conditions such as tissue repair, inflammation, fibrosis and carcinogenesis (Powell et al., 2011). We have recently demonstrated that intestinal mesenchymal-specific signals play important in vivo physiological roles in intestinal inflammation and carcinogenesis (Koliaraki et al., 2012; Roulis et al., 2014; Koliaraki et al., 2015). Here we describe the enzymatic method used for the isolation and culture of mesenchymal cells ...
[摘要] 在过去20年间，肠间质细胞（IMC）已经作为重要的细胞类型出现，通过提供结构支持和生长调节元件在肠发育和体内平衡中起着中心作用。 IMC还积极参与伤口愈合反应，从而调节病理状况，例如组织修复，炎症，纤维化和癌发生（Powell等人，2011）。 我们最近已经证明肠间充质特异性信号在肠炎症和癌发生中在体内起重要的生理作用（Koliaraki等人，2012; Roulis等人，/em>。，2014; Koliaraki 。。，2015）。 在这里我们描述了用于从成年小鼠肠道分离和培养间充质细胞的酶法。
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