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Link to original content: http://www.ncbi.nlm.nih.gov/pubmed/23408158
Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis - PubMed Skip to main page content
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. 2012 Dec 15;1(12):1273-6.
doi: 10.1242/bio.20123228. Epub 2012 Oct 23.

Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis

Shoko Ishibashi  1 , Rebecca Cliffe, Enrique Amaya
Affiliations

Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis

Shoko Ishibashi et al. Biol Open. .

Abstract

In the past decade, Xenopus tropicalis has emerged as a powerful new amphibian genetic model system, which offers all of the experimental advantages of its larger cousin, Xenopus laevis. Here we investigated the efficiency of transcription activator-like effector nucleases (TALENs) for generating targeted mutations in endogenous genes in X. tropicalis. For our analysis we targeted the tyrosinase (oculocutaneous albinism IA) (tyr) gene, which is required for the production of skin pigments, such as melanin. We injected mRNA encoding TALENs targeting the first exon of the tyr gene into two-cell-stage embryos. Surprisingly, we found that over 90% of the founder animals developed either partial or full albinism, suggesting that the TALENs induced bi-allelic mutations in the tyr gene at very high frequency in the F0 animals. Furthermore, mutations tyr gene were efficiently transmitted into the F1 progeny, as evidenced by the generation of albino offspring. These findings have far reaching implications in our quest to develop efficient reverse genetic approaches in this emerging amphibian model.

Keywords: Nuclease; Reverse genetics; TAL effector; Targeted mutagenesis.

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Conflict of interest statement

Competing interests: The authors have no competing interests to declare.

Figures

Fig. 1.
Fig. 1.. Mutations in the tyr gene following TALEN mRNAs injection into embryos.
(A) TALEN target sequences in exon 1, 27 bp downstream from the initiation codon in the tyr gene are boxed in yellow. (B) Mutations induced by TALENs. 400 pg of each TALEN RNA was injected into 2-cell stage embryos and DNA was extracted at stage 18 (mid-neurula) from six individual embryos. The wild type sequence is shown at the top with TALEN binding sites marked in yellow. Sequences from each individual embryo is shown separately, with each having a separate annotation, from “a” to “f”. Deletions are indicated by dashes. The sizes of deletion (▵) and insertion (+) are presented in the right of the each allele. The number of times that each mutant allele was isolated is shown between square brackets.
Fig. 2.
Fig. 2.. Phenotypic analysis of the embryos injected with tyr TALEN RNAs.
(A) Representative embryos injected with tyr TALEN RNAs. We screened the injected embryos and split them into groups showing wild type, partial albinism or full albinism. (B) Number of embryos showing wild type, partial or full albinism after injecting with mRNA encoding the shown TALENs.
Fig. 3.
Fig. 3.. Phenotype in F0 tadpoles and adult frogs, with bi-allelic mutations in the tyr gene, resulting in albinism.
(A) Tadpole injected with 600 pg of RNA encoding Left TALEN (upper) and albino tadpole injected with 300 pg of RNA encoding each of the TALEN pair targeting the tyr gene (lower). Scale bar: 1 mm. (B) Control, wild type (right) and albino frog (left), generated by injecting TALEN mRNAs targeting the tyr gene.
Fig. 4.
Fig. 4.. Mutations induced by TALENs are heritable through the germline.
(A) F1 cleavage stage embryos produced by mating F0 frogs previously injected with mRNAs encoding TALENs targeting the tyr gene, give rise to a mixture of albino and pigmented embryos. (B) F1 tadpoles produced by mating F0 frogs previously injected with mRNAs encoding TALENs targeting the tyr gene, give rise to a mixture of albino tadpoles, flanking a pigmented sibling tadpoles (centre). (C) The tyr loci were amplified by PCR from three randomly selected F1 embryos (F1-1 to 3), and PCR clones from these embryos were sequenced. One was homozygous wild type (top), the other two were heterozygous mutant. F1-a represents sequence from an albino F1 embryo, which confirmed that the embryo was homozygous mutant. Genotype and frequency of sequence are shown in the right of the each allele. Scale bar: 1 mm.
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