Efficient CRISPR/Cas9-Mediated Mutagenesis in Primary Murine T Lymphocytes.
Efficient CRISPR/Cas9-Mediated Mutagenesis in Primary Murine T Lymphocytes.
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DOI：
10.1002/cpim.62
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发表时间：
2019-03
影响因子：
--
通讯作者：
Schwartzberg PL
中科院分区：
文献类型：
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作者：
Huang B;Johansen KH;Schwartzberg PL
直接改变T淋巴细胞基因表达的能力为理解T细胞的生物学、信号和功能提供了强有力的工具。对T细胞克隆和原代T细胞的操作主要是通过分别使用cDNA或shRNA的过度表达或基因沉默研究来完成的，这些cDNA或shRNA通常通过逆转录病毒或慢病毒转导或直接转染法传递。基于CRISPR/Cas9的突变技术的最新发展使基因组编辑发生了革命性的变化，使得对许多细胞类型的前所未有的基因操作变得更精确和更容易。在这一章中，我们概述了CRISPR/Cas9介导的诱变方案，利用逆转录病毒传递引导RNA对Cas9转基因小鼠的原代T淋巴细胞进行诱变。
The ability to alter gene expression directly in T lymphocytes has provided a powerful tool for understanding T cell biology, signaling and function. Manipulation of T cell clones and primary T cells has been accomplished primarily through overexpression or gene-silencing studies using cDNAs or shRNAs, respectively, which are often delivered by retroviral or lentiviral transduction or direct transfection methods. The recent development of CRISPR/Cas9-based mutagenesis has revolutionized genomic editing, allowing unprecedented genetic manipulation on many cell types with greater precision and ease. In this chapter, we outline a protocol for CRISPR/Cas9-mediated mutagenesis in primary T lymphocytes from Cas9 transgenic mice using retroviral delivery of guide RNAs.