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Anti-rat IgG, HRP-linked Antibody

Company: Cell Signaling Technology
Catalog#: 7077
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Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9
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2017-04-05
[Abstract]  CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which ... [摘要]  基于CRISPR-Cas9的敲除策略越来越多地用于分析基因功能。然而,生物信号通路中的冗余和重叠功能可能需要产生多基因敲除细胞,这仍然是一个相对费力的过程。在这里,我们详细介绍了多色LentiCRISPR载体在人体细胞中同时产生单次和多次敲除的应用。我们提供了一个完整的方案,包括指导RNA设计,LentiCRISPR克隆,病毒生产和转导,以及排序和筛选敲除细胞的策略。该过程的有效性通过同时删除白血病细胞系中多达四个程序性细胞死亡介质和来自患者来源的急性淋巴细胞白血病异种移植物,其中单细胞克隆是不可行的。该协议允许任何具有基本细胞生物学设备的实验室,生物安全2级设备和荧光激活细胞分选功能,可在一个月内有效产生单基因和多基因敲除细胞系或原代细胞。

从对细菌基因组中被称为聚簇定期交织的短回文重复(CRISPR)的遗传元件的好奇的初步观察开始(Ishino等人,1987; Mojica等人,2000 )和随后在哺乳动物细胞中的基因编辑(Cong等人,2013; Mali等人,2013),CRISPR-Cas9已经成为廉价和有效的基因编辑。随着从烟草植物细胞到斑马鱼和原代人类细胞(Hsu等人,2014)的细胞系统的成功应用,CRISPR-Cas9可以通过短的20个核苷酸RNA序列的设计来引导在大基因组内的靶向DNA双链断裂(DSB)(Park等人,2016)。 ...

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