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Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells.
优化 RNP 转染,用于原代 T 细胞中高效的 CRISPR/Cas9-mediated 基因敲除。

摘要

CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell-based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will greatly extend the feasibly of target gene discovery and validation in primary T cells and simplify the gene editing process for next-generation immunotherapies.

译文

CRISPR (聚集的、规则间隔的、短回文重复序列)/Cas9 (CRISPR 相关蛋白 9) 已经成为在各种物种中产生基因敲除的首选工具。原代 T 细胞中高效基因编辑的能力不仅代表了研究基因功能的一个有价值的研究工具,也为基于 T 细胞的免疫治疗带来了巨大的希望, 如下一代嵌合抗原受体 (CAR) T 细胞。先前尝试将 CRIPSR/Cas9 应用于原代 T 细胞的基因编辑已经导致了高度可变的敲除效率和所需的 T 细胞受体 (TCR) 刺激, 因此很大程度上排除了对参与 T 细胞活化或分化的基因的研究。在这里,我们描述了一种优化的方法,在没有 TCR 刺激的情况下,Cas9/RNP 转染原代小鼠和人类 T 细胞,这导致在群体水平上几乎完全失去目标基因表达, 减轻选择的需要。我们相信,这种方法将极大地扩展目标基因发现和在原代 T 细胞中验证的可行性,并简化下一代免疫治疗的基因编辑过程。

transfection

肿瘤 转化 实验技术
概述  :  

转染采用与质粒DNA转化受体细胞相似的方法,即宿主菌先经过CaCl2,电穿孔等处理成感受态细菌,再将重组噬菌体DNA直接导入受体细胞,进入感受态细菌的噬菌体DNA可以同样复制和繁殖,这种方式称为转染。 转染是转化的一种特殊形式。常规转染技术分为两大类,一类是瞬时转染,一类是稳定转染(永久转染)。前者外源DNA/RNA不整合到宿主染色体中,因此一个宿主细胞中可存在多个拷贝数,产生高水平的表达,但通常只持续几天,多用于启动子和其它调控元件的分析。一般来说,超

Transfection   /trænz'fɛkʃən/

释    义   n. [遗] 转染

例    句   However, they found that RNA transfection poses a significant challenge: When added to mature human skin cells, mRNA provokes an immune response meant to defend against viruses made of RNA. 但是,他们发现,用RNA转染面临着巨大的挑战:当将mRNA添加到成熟的人体皮肤细胞时,信使RNA产生了强烈的免疫反应来排斥RNA病毒。

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