| A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9
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Author:
Date:
2017-06-05
[Abstract] The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific target sequence. One drawback of this system is that the production of sgRNA in the laboratory is laborious since it requires cloning of an sgRNA sequence, in vitro transcription reaction and sgRNA purification. An alternative to targeting Cas9 activity by sgRNA is to target it with two small RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Both of these small RNAs can be chemically synthesized which makes the production of ...
[摘要] 聚类规则间隔短回文重复（CRISPR）/ CRISPR相关蛋白9（Cas9）系统是使用最广泛的基因组编辑工具。一个常见的CRISPR / Cas9系统由两个组成部分组成：单导RNA（sgRNA）和Cas9。在特定靶序列引入双链断裂（DSB）需要两种成分。该系统的一个缺点是实验室中sgRNA的生产是费力的，因为它需要在体外转录反应和sgRNA纯化之间克隆sgRNA序列。通过sgRNA靶向Cas9活性的替代方案是用两种小RNA：CRISPR RNA（crRNA）和反式激活性crRNA（tracrRNA）进行靶向。这两种小RNA可以化学合成，这使得与sgRNA相比，这些RNA的产生不那么困难。 CRISPR / Cas9系统的另一个缺点是已经报告了脱靶效应。然而，已经开发了改进形式的Cas9以最小化离靶效应。例如，仅当两个引导RNA在规定的距离内结合相对的链时，切口酶型Cas9（nCas9）和FokI结构域融合的催化无活性的Cas9（FokI-dCas9; fCas9）才诱导DSB。在本协议中，我们描述了使用结合crRNA，tracrRNA和Cas9修饰形式的CRISPR / Cas9系统来生产突变小鼠的实验系统。该方法不仅有利于制备用于基因组编辑系统的试剂，而且可以降低脱靶效应的风险。
背景 ...
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| Establishing Primary Malignant Pleural Mesothelioma (MPM) Cell Cultures
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Author:
Date:
2012-11-05
[Abstract] This is a general protocol for the isolation and maintenance of primary MPM cultures as a tool for the identification of Tumor Initiating Cells and early progenitor-targeting drugs (Cioce et al., 2010). Primary cultures can be propagated efficiently for 8-12 weeks and xenotransplanted in NOD/SCID mice while retaining the histofeatures of the originating tumor (Canino et al., 2012). The protocol is suitable for both MPM solid specimens and pleural effusion. For increased clarity, initially two separate sections addressing the isolation of MPM cells from solid tumors and pleural effusions are here provided.
[摘要] 这是用于分离和维持初级MPM培养物的一般方案，作为鉴定肿瘤启动细胞和早期祖细胞靶向药物的工具（Cioce等人，2010）。 原代培养物可以有效繁殖8-12周，并且在NOD/SCID小鼠中异种移植，同时保留起源肿瘤的组织特征（Canino等人，2012）。 该方案适用于MPM固体标本和胸腔积液。 为了增加清晰度，最初提供了两个分开的部分，其涉及从实体瘤和胸腔积液中分离MPM细胞。
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