利用转录激活样效应核酸酶（TALENs）对多能细胞进行基因组修饰

Genome Modification of Pluripotent Cells by Using Transcription Activator-Like Effector Nucleases (TALENs).

作者信息

Taheri-Ghahfarokhi Amir, Malaver-Ortega Luis F, Sumer Huseyin

机构信息

Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran.

Monash Institute for Medical Research, Monash University, Clayton, VIC, 3168, Australia.

出版信息

Methods Mol Biol. 2015;1330:253-67. doi: 10.1007/978-1-4939-2848-4_19.

DOI:10.1007/978-1-4939-2848-4_19

PMID:26621602

Abstract

Interest is increasing in transcription activator-like effector nucleases (TALENs) as a tool to introduce targeted double-strand breaks into the large genomes of human and animal cell lines. The produced DNA lesions stimulate DNA repair pathways, error-prone but dominant non-homologous end joining (NHEJ) and accurate but less occurring homology-directed repair (HDR), and as a result targeted genes can be modified. Here, we describe a modified Golden-Gate cloning method for generating TALENs and also details for targeting genes in mouse embryonic stem cells. The protocol described here can be used for modifying the genome of a broad range of pluripotent cell lines.

摘要

作为一种将靶向双链断裂引入人类和动物细胞系大基因组的工具，转录激活样效应核酸酶（TALENs）正受到越来越多的关注。产生的DNA损伤会刺激DNA修复途径，即易出错但占主导的非同源末端连接（NHEJ）和精确但较少发生的同源定向修复（HDR），结果是靶向基因可以被修饰。在这里，我们描述了一种用于生成TALENs的改良金门克隆方法，以及在小鼠胚胎干细胞中靶向基因的详细步骤。这里描述的方案可用于修饰多种多能细胞系的基因组。