RRC ID 47703
Author Ansai S, Inohaya K, Yoshiura Y, Schartl M, Uemura N, Takahashi R, Kinoshita M.
Title Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator-like effector nucleases in medaka.
Journal Dev Growth Differ
Abstract Genome editing using engineered nucleases such as transcription activator-like effector nucleases (TALENs) has become a powerful technology for reverse genetics. In this study, we have described efficient detection methods for TALEN-induced mutations at endogenous loci and presented guidelines of TALEN design for efficient targeted mutagenesis in medaka, Oryzias latipes. We performed a heteroduplex mobility assay (HMA) using an automated microchip electrophoresis system, which is a simple and high-throughput method for evaluation of in vivo activity of TALENs and for genotyping mutant fish of F1 or later generations. We found that a specific pattern of mutations is dominant for TALENs harboring several base pairs of homologous sequences in target sequence. Furthermore, we found that a 5' T, upstream of each TALEN-binding sequence, is not essential for genomic DNA cleavage. Our findings provide information that expands the potential of TALENs and other engineered nucleases as tools for targeted genome editing in a wide range of organisms, including medaka.
Volume 56(1)
Pages 98-107
Published 2014-1-1
DOI 10.1111/dgd.12104
PMID 24286287
MeSH Animals Endodeoxyribonucleases / genetics Endodeoxyribonucleases / metabolism* Gene Targeting / methods* Heteroduplex Analysis Mutagenesis, Site-Directed / methods* Oryzias / genetics*
IF 1.723
Times Cited 41
WOS Category DEVELOPMENTAL BIOLOGY CELL BIOLOGY
Resource
Medaka d-rR/TOKYO (MT837) OK-Cab (MT830) DJ-1(delta7) (MT972) MC4R(delta7) (MT973) MC4R(delta11) (MT974) Sakyo (WS1164)