期刊
METHODS
卷 69, 期 1, 页码 2-8出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2014.02.013
关键词
TALENs; Rice; Targeted mutagenesis; Gene knockout
资金
- Ministry of Agriculture of China [2013ZX08002-004, 2013ZX08010-002]
- National Natural Science Foundation of China [201263, 383601, 31200273]
Targeted gene mutagenesis is a powerful tool for elucidating gene function and facilitating genetic improvement in rice. TALENs (transcription activator-like effector nucleases), consisting of a custom TALE DNA binding domain fused to a nonspecific Fokl cleavage domain, are one of the most efficient genome engineering methods developed to date. The technology of TALENs allows DNA double-strand breaks (DSBs) to be introduced into predetermined chromosomal loci. DSBs trigger DNA repair mechanisms and can result in loss of gene function by error-prone non-homologous end joining (NHEJ), or they can be exploited to modify gene function or activity by precise homologous recombination (HR). In this paper, we describe a detailed protocol for constructing TALEN expression vectors, assessing nuclease activities in vivo using rice protoplast-based assays, generating and introducing TALEN DNAs into embryogenic calluses of rice and identifying TALEN-generated mutations at targeted genomic sites. Using these methods, To rice plants resulting from TALEN mutagenesis can be produced within 4-5 months. (C) 2014 Elsevier Inc. All rights reserved.
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