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Dev Growth Differ
2014 Jan 01;561:108-14. doi: 10.1111/dgd.12105.
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Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases.
Sakane Y
,
Sakuma T
,
Kashiwagi K
,
Kashiwagi A
,
Yamamoto T
,
Suzuki KT
.
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Transcription activator-like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X. laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues (tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40-80%. Next, we show early introduction of TALEN-mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X. laevis.
Figure 1. Phenotypes of the tyr/egfp transcription activator-like effector nuclease (TALEN)-injected embryos. Bright (upper) and fluorescence (lower) images show pigmentation and green fluorescent protein (GFP) fluorescence, respectively. Uninjected, uninjected embryos. tyr/egfp TALENs, embryos co-injected with each pair of TALEN mRNAs targeting tyr and egfp. Genomic DNA of the uninjected (an asterisk) and the TALEN-injected (i, ii, and iii) embryos were subjected to heteroduplex mobility assay (HMA), restriction fragment length polymorphism (RFLP) analysis, and DNA sequencing (see Fig. 3).
Figure 2. Phenotype frequency and toxicity of the tyr/egfp transcription activator-like effector nuclease (TALEN)-injected embryos. (A) Percentage of phenotypes in the uninjected embryos (Uninjected) and the tyr/egfp TALEN-injected embryos (tyr/egfp TALENs). Except for abnormally developed embryos, phenotypes were divided into four groups (severe, moderate, weak, and normal) according to the extent of loss of pigments in the RPE. Total numbers of individuals are shown at the top of each graph. (B) Correlation between albinism and loss of green fluorescent protein (GFP) fluorescence in the TALEN-injected embryos. Corresponding images in each embryo show phenotypes of pigmentation (severe, moderate, weak, and normal; upper images) and GFP fluorescence (lower images).
Figure 4. Timing of introduction of mutations after transcription activator-like effector nucleases (TALENs) mRNA injection. TALEN-mediated mutations in tyra, tyrb, and egfp at the early stages of embryogenesis were detected by heteroduplex mobility assay (HMA) (upper) and restriction fragment length polymorphism (RFLP) analysis (lower). Genomic DNA was extracted from 5 to 10 embryos at the stages indicated at the top of the gel electrophoresis images. Arrows, open and closed arrowheads are described in Figure 3A. C and T indicate the uninjected embryos and the TALEN-injected embryos, respectively.
