| Selective Isolation of Retroviruses from Extracellular Vesicles by Intact Virion Immunoprecipitation
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Author:
Date:
2018-09-05
[Abstract] There exists a wide variety of techniques to isolate and purify viral particles from cell culture supernatants. However, these techniques vary greatly in ease of use, purity, yield and impact on viral structural integrity. Most importantly, it is becoming evident that secreted extracellular vesicles (EVs) co-purify with retroviruses using nearly all purification methods due to nearly indistinguishable biophysical characteristics such as size, buoyant density and nucleic acid content. Recently, our group has illustrated a means of isolating intact and highly enriched retroviral virions from EV-containing cell supernatants using an immunoprecipitation approach targeting the viral envelope glycoprotein of the Moloney Murine Leukemia Virus (Renner et al., 2018). This technique, that ...
[摘要] 存在多种从细胞培养上清液中分离和纯化病毒颗粒的技术。然而，这些技术在易用性，纯度，产量和对病毒结构完整性的影响方面差异很大。最重要的是，由于几乎无法区分的生物物理特征，例如大小，浮力密度和核酸含量，使用几乎所有纯化方法分泌的细胞外囊泡（EV）与逆转录病毒共同纯化变得明显。最近，我们小组已经阐明了一种利用针对Moloney鼠白血病病毒的病毒包膜糖蛋白的免疫沉淀方法从含有EV的细胞上清液中分离完整和高度富集的逆转录病毒病毒颗粒的方法（Renner et al。， 2018）。这种技术，我们称之为完整的病毒粒子免疫沉淀（IVIP），使我们能够表征这些逆转录病毒表面上表位的可及性，并评估病毒包膜中病毒编码的整合膜蛋白Glycogag（gPr80）的方向。该方案的正确实施使得能够快速，简单且可重复地制备完整且高度纯化的逆转录病毒颗粒，其没有可检测的EV污染物。
【背景】广泛使用的分离逆转录病毒的方法，如人类免疫缺陷病毒（HIV）和小鼠白血病病毒（MLV），包括沉淀，色谱，超滤，超速离心，以及各种其他粒子分离方法（评论在Nestola et al。，2015）。虽然每种技术都有其特定的优点，缺点和局限性，但所有方法的共同关注点是细胞分泌的细胞外囊泡（EV）的共同纯化。
EV构成由所有细胞类型分泌的膜衍生囊泡的异质群体（Yanez-Mo ...
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| Active Cdk5 Immunoprecipitation and Kinase Assay
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Author:
Date:
2017-07-05
[Abstract] Cdk5 activity is regulated by the amounts of two activator proteins, p35 and p39 (Tsai et al., 1994; Zheng et al., 1998; Humbert et al., 2000). The p35-Cdk5 and p39-Cdk5 complexes have differing sensitivity to salt and detergent concentrations (Hisanaga and Saito, 2003; Sato et al., 2007; Yamada et al., 2007; Asada et al., 2008). Cdk5 activation can be directly measured by immunoprecipitation of Cdk5 with its bound activator, followed by a Cdk5 kinase assay. In this protocol, buffers for cell lysis and immunoprecipitation are intended to preserve both p35- and p39-Cdk5 complexes to assess total Cdk5 activity. Cells are lysed and protein concentration is determined in the post-nuclear supernatant. Cdk5 is immunoprecipitated from equal ...
[摘要] Cdk5活性受两种激活蛋白p35和p39（Tsai et al。，1994; Zheng et al。，1998; Humbert等人）的量的调节，2000）。 p35-Cdk5和p39-Cdk5复合物对盐和洗涤剂浓度的敏感性不同（Hisanaga和Saito，2003; Sato et al。，2007; Yamada等人， 2007; Asada 等人，2008）。 Cdk5激活可以通过Cdk5与其结合的激活剂的免疫沉淀直接测量，随后进行Cdk5激酶测定。在该方案中，用于细胞裂解和免疫沉淀的缓冲液旨在保持p35-和p39-Cdk5复合物以评估总Cdk5活性。裂解细胞，并在核后上清液中测定蛋白浓度。 Cdk5在实验组之间从等量的总蛋白免疫沉淀。然后进行洗涤以除去外来蛋白质并平衡激酶缓冲液中的Cdk5-活化剂复合物。然后将Cdk5与组蛋白H1孵育，组蛋白H1是Cdk5和[γ- 32 P] ATP在体外成功建立的靶标。反应通过SDS-PAGE解析并转移到膜上，用于可视化H1磷酸化和免疫沉淀的Cdk5水平的免疫印迹。我们已经使用该测定来建立p39作为少突神经胶质谱系中Cdk5的主要活化剂。然而，该测定法适用于对裂解条件进行适当调整的其它细胞谱系或组织。
【背景】虽然Cdk5通常与神经元功能相关，但最近的工作已经证明Cdk5也可以调节少突胶质细胞祖细胞（OPC）的发育（Tang等人，1998; ...
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| Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina
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Author:
Date:
2017-02-05
[Abstract] All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7-10 days in vitro. During ...
[摘要] 构成成熟视网膜的所有七种视网膜细胞类型都是由普通的多能视网膜祖细胞池（RPC）产生的（Wallace，2011）。已经产生足够数量的特定细胞类型的RPC知道的一种方式是通过负反馈信号，其由分化细胞发射并且必须达到阈值水平以阻止该细胞类型的额外分化。评估负反馈信号是否由分化细胞发出的关键测定是异源沉淀测定，其中早期RPC被解离并用BrdU标记，然后与20倍过量的解离的分化细胞混合。然后将组合的细胞再次聚集并在细胞膜上培养7-10天。在这段时间内，RPC将会分化，BrdU + RPC的命运可以使用细胞类型特异性标记进行评估。开发这种沉淀测定的研究人员最初表明，当两种细胞类型混合在一起时，新生儿RPC与胚胎RPC相比，在加速进度条件下产生杆（Watanabe和Raff，1990; Watanabe等，1997）。我们已经使用这种测定来证明我们发现作为视网膜神经节细胞（RGC）分化的负调节物的声刺猬（Shh）促进RPC增殖（Jensen和Wallace，1997; Ringuette等，2014）。最近我们修改了异质性沉淀测定法，以评估视网膜无长突细胞的反馈信号的作用，将转化生长因子β2（Tgfβ2）鉴定为负反馈信号，并将Pten作为Tgfβ2应答的调节剂（Ma et al。，2007 ; ...
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