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1 June 2013 Targeted Gene Disruption in the Xenopus tropicalis Genome using Designed TALE Nucleases
Keisuke Nakajima, Yuya Nakai, Morihiro Okada, Yoshio Yaoita
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Abstract

Transcription activator-like effector nucleases (TALENs) are attractive and powerful molecular tools for targeted gene disruption because of their simple design and quick assembly. To evaluate the utility of TALENs in genome editing in Xenopus tropicalis, we prepared nine pairs of TALENs for the tyrosinase, noggin and MMP-9TH genes. All of the TALENs had some activity in a single-strand annealing assay using a cultured frog cell line, suggesting double-stranded DNA cleavage activity by the TALENs at their target site. The injection of mRNAs encoding TALENs into fertilized X. tropicalis embryos resulted in Cel-1 cleavage of the PCR fragment containing the target site amplified from embryo genomic DNA, indicating that a mutation in the target gene had occurred during embryogenesis. These mutations were confirmed by the sequencing of clones derived from the PCR fragments of genomic DNA. Patches of vitiligo were observed in tadpoles raised from fertilized eggs that had been injected with mRNAs of TALENs for the tyrosinase gene. TALENs containing the repeat variable di-residue (RVD) NN appeared to show more activity than TALENs containing RVD NK, although both RVD NN and NK preferentially associate with a G nucleotide.

© 2013 Zoological Society of Japan
Keisuke Nakajima, Yuya Nakai, Morihiro Okada, and Yoshio Yaoita "Targeted Gene Disruption in the Xenopus tropicalis Genome using Designed TALE Nucleases," Zoological Science 30(6), 455-460, (1 June 2013). https://doi.org/10.2108/zsj.30.455
Received: 16 August 2012; Accepted: 1 December 2012; Published: 1 June 2013
JOURNAL ARTICLE
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KEYWORDS
gene knockout
Gene targeting
genome edition
TALENs
Xenopus tropicalis
ZFNs
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