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Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.
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Fig 1. Injection of TALEN mRNAs into GV oocytes allows efficient tyrosinase gene disruption.
(A) Schematic diagram of the experiment to express TALENs in Xenopus laevis eggs before fertilization. (B) Development of tyrosinase (tyr) TALENs-expressed oocytes. TALEN mRNA-injected GV oocytes, followed by in vitro maturation and ICSI, were able to develop to the swimming tadpole stage although mRNA injection itself seems to decrease the developmental capacity of in vitro matured eggs. Actual numbers of embryos that were injected with sperm and that developed to each developmental stages are indicated next to the corresponding bars. (C-E) Expression of tyr TALENs in GV oocytes allowed almost complete albino phenotypes in F0 embryos and frogs. Enlarged pictures of two TALEN-RL-expressed tadpoles are shown in Fig 1D. Control R represents control embryos and frogs in which only right side TALEN was expressed.
doi:10.1371/journal.pone.0142946.g001
Fig 2. Phenotypes of embryos that were injected with pax6 TALEN mRNAs.
(A) Injection of pax6 TALEN mRNAs to GV oocytes, followed by in vitro maturation and ICSI, recapitulated the null mutant phenotype of pax6 in F0 embryos. Examples of knock-out tadpoles with different magnifications (a-f; pax6 TALEN-expressed tadpoles, g-l; control tadpoles) are shown. The percentages of embryos that showed the knock-out phenotype of pax6 are summarized in the graph, as judged by the degree of eye deformation. Embryos without TALEN mRNA injection are used as a control (Non injected). (B) Different phenotypes are observed at the tadpole stage between conventional embryo injection (Fig 2B) and the oocyte injection method (Fig 2A). TALEN mRNAs were injected into fertilized one-cell stage embryos. Examples of pax6 TALEN-expressed tadpoles with different magnifications (a-d; pax6 TALEN-expressed tadpoles, e-h; control tadpoles) are shown.
doi:10.1371/journal.pone.0142946.g002
Fig 3. Double knock-out of pax6 and tyr in Xenopus F0 embryos.
(A) Double knock-out of pax6 and tyr was achieved by TALEN mRNA injection to GV oocytes. A double knock-out tadpole is shown. (B) Detection of pax6 and tyr mutants by RFLP analysis. Wild type embryos are cut by restriction enzymes (Ui: uninjected embryo), while mutant embryos are not digested. Digested PCR products appear at lower bands (marked by open arrowheads) and the undigested is marked by closed arrowheads. M represents 100 bp ladder marker. (C) Sequencing of three different embryos (#3, #6, #9; the numbers correspond to those in Fig 3B) revealed the 100% mutation rate in pax6 and tyr double knock-out embryos.
doi:10.1371/journal.pone.0142946.g003
Fig 4. The deletion of a large genetic locus is achieved by the expression of two sets of TALENs by oocyte injection.
(A) TALENs were designed to disrupt one of the exons of mars2-like (mars2-l) gene. PCR primers were used to confirm gene knock-out. Exons are marked by boxes. (B) The injection of TALEN mRNAs into GV oocytes (blue bar), but not into fertilized embryos (red bar), showed better knock-out of the mars2-l exon, as revealed by qPCR analyses. Control represents embryos injected only with right TALEN mRNA. N = 3–4 independent experiments. Error bars are standard errors. *P < 0.05. **P < 0.01. (C) Expression of TALENs before fertilization allowed the production of an embryo carrying only the deleted mars2-l exon in the F0 generation (lane 3, 7 and 8 in TALEN B-D, red color). Genomic DNA was extracted from single embryos at St. 10.5–11 and subjected to PCR analysis. M represents 1.5 kp and 100 bp ladder marker. (D) Large deletions of exon 2 in mars-l were confirmed by sequencing. Sample numbers correspond to those in Fig 4C. A part of inverted mars-l sequence was inserted in sample #3.
doi:10.1371/journal.pone.0142946.g004
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