| Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells
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Author:
Date:
2017-02-05
[Abstract] A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden et al., 2015).
[摘要] 合成的致死相互作用是一种遗传相互作用，其中两种基因之一的破坏单独具有影响，但它们的组合破坏是致命的。有关合成致死相互作用的知识可以帮助阐明网络结构和确定人类疾病如癌症的候选药物靶标。在果蝇中，组合基因破坏已经通过组合多个RNAi试剂而实现。这里我们通过组合CRISPR和RNAi来描述高通量组合基因破坏的协议。这种方法以前导致了高度可重现和保守的合成致死相互作用的识别（Housden等人，2015）。
背景 遗传相互作用的知识，如合成致死性，对于确定基因之间的功能关系可能是无价的。例如，酵母中的大规模遗传相互作用屏幕最近被用于组装全球“细胞功能的接线图”（Costanzo等人，2016）。或者，可以使用特定类型的遗传相互作用，例如合成致死相互作用来鉴定包括癌症在内的疾病的药物靶标（Kaelin，2005）。
＆nbsp;鉴定合成相互作用需要组合破坏两个基因。以前在果蝇细胞培养中实现该方法的方法是同时递送多个dsRNA试剂（例如，Fisher等人，2015）。然而，RNAi试剂具有局限性，包括脱靶效应和不完全靶标敲低，当多个试剂一起递送时，RNAi试剂复合。通过将CRISPR诱变与单个dsRNA处理相结合，可以避免这些问题，从而更简单地解释筛选结果并强化“点击”识别。
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| Differentiation of Human Embryonic Stem Cells into Cone Photoreceptors
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Author:
Date:
2016-07-20
[Abstract] Photoreceptors are specialized retinal neurons able to respond to light in order to generate visual information. Among photoreceptors, cones are involved in colors discrimination and high-resolution central vision and are selectively depleted in macular degenerations and cone dystrophies. A possible therapeutic solution for these disorders is to replace degenerating cells with functional cones. Here, we describe a simple protocol for the rapid production of large amount of cone photoreceptors from human pluripotent stem cells. The differentiation protocol is based on the “default pathway” of neural induction using the BMP, TGFβ and WNT antagonist COCO.
[摘要] 光感受器是能够响应光以产生视觉信息的专门的视网膜神经元。 在光感受器中，视锥细胞参与颜色辨别和高分辨率中心视觉，并且选择性缺失黄斑变性和视锥营养不良。 这些疾病的可能的治疗溶液是用功能性锥体代替变性细胞。 在这里，我们描述了一个简单的协议，从人类多能干细胞快速生产大量的锥形感光细胞。 分化方案基于使用BMP，TGFβ和WNT拮抗剂COCO的神经诱导的"默认途径"。
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| Glioma Associated Stem Cells (GASCs) Isolation and Culture
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Author:
Date:
2015-05-05
[Abstract] Glioma Associated Stem Cells (GASCs) represent a population of non-tumorigenic multipotent stem cells hosted in the microenvironment of human gliomas. In vitro, these cells are able, through the release of exosomes, to increase the biological aggressiveness of glioma-initiating cells. The clinical importance of this finding is supported by the strong prognostic value associated with the GASCs surface immunophenotype thus suggesting that this patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma (Bourkoula et al., 2014).
[摘要] 胶质瘤相关干细胞（GASC）代表在人类神经胶质瘤的微环境中存在的非致瘤性多能干细胞群体。 在体外，这些细胞能够通过外来体的释放增加胶质瘤起始细胞的生物攻击性。 该发现的临床重要性得到与GASCs表面免疫表型相关的强预后价值的支持，因此表明这种基于患者的方法可以提供开创性的方法来预测预后并开发靶向肿瘤基质的新策略（Bourkoula等人， et al。，2014）。
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