Monoclonal Cell Line Generation and CRISPR/Cas9 Manipulation via Single‐Cell Electroporation,Small

当前位置： X-MOL 学术 › Small › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Monoclonal Cell Line Generation and CRISPR/Cas9 Manipulation via Single‐Cell Electroporation
Small ( IF 13.0 ) Pub Date : 2018-02-12 , DOI: 10.1002/smll.201702495
Ruiguo Yang _{1,

2} , Vincent Lemaître ₂ , Changjin Huang _{1,

2} , Abbas Haddadi _{2,

3} , Rebecca McNaughton ₂ , Horacio D Espinosa _{1,

2,

4}

Affiliation

Stably transfected cell lines are widely used in drug discovery and biological research to produce recombinant proteins. Generation of these cell lines requires the isolation of multiple clones, using time‐consuming dilution methods, to evaluate the expression levels of the gene of interest. A new and efficient method is described for the generation of monoclonal cell lines, without the need for dilution cloning. In this new method, arrays of patterned cell colonies and single cell transfection are employed to deliver a plasmid coding for a reporter gene and conferring resistance to an antibiotic. Using a nanofountain probe electroporation system, probe positioning is achieved through a micromanipulator with sub‐micron resolution and resistance‐based feedback control. The array of patterned cell colonies allows for rapid selection of numerous stably transfected clonal cell lines located on the same culture well, conferring a significant advantage over slower and labor‐intensive traditional methods. In addition to plasmid integration, this methodology can be seamlessly combined with CRISPR/Cas9 gene editing, paving the way for advanced cell engineering.

中文翻译：

通过单细胞电穿孔生成单克隆细胞系和 CRISPR/Cas9 操作

稳定转染的细胞系广泛用于药物发现和生物研究以生产重组蛋白。这些细胞系的产生需要使用耗时的稀释方法分离多个克隆，以评估感兴趣基因的表达水平。描述了一种用于产生单克隆细胞系的新且有效的方法，无需稀释克隆。在这种新方法中，采用图案化细胞集落阵列和单细胞转染来传递编码报告基因并赋予抗生素抗性的质粒。使用纳米喷泉探针电穿孔系统，通过具有亚微米分辨率和基于电阻的反馈控制的显微操纵器实现探针定位。图案化细胞集落阵列允许快速选择位于同一培养孔上的大量稳定转染的克隆细胞系，与速度较慢且劳动密集型的传统方法相比具有显着优势。除了质粒整合之外，该方法还可以与 CRISPR/Cas9 基因编辑无缝结合，为先进的细胞工程铺平道路。

更新日期：2018-02-12

点击分享查看原文

点击收藏

公开下载

阅读更多本刊最新论文本刊介绍/投稿指南11