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Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3'UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3'UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf). In contrast, TALEN mRNAs without this 3'UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT) stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.
Fig. 1. Schematic representation of the experimental procedure.
Fig. 2. Time course of mutations induced by microinjection with either Tyr-TALEN or Tyr-TALEN-DS mRNAs.
Oocytes (squares) or fertilized eggs (circles) were injected with either Tyr-TALEN (open squares and circles) or Tyr-TALEN-DS (closed squares and a circle) mRNAs. The injected oocytes were treated with progesterone, transferred into a host female, and fertilized. The genomic DNA was extracted from four to six individual embryos at the indicated times. The target DNA was amplified by PCR and cloned. The DNA sequence was determined for five to fifteen clones per embryo (supplementary material Figs S1, S2). The data are expressed as the mean value ± standard error. The abscissa is indicated by hours (A) and days (B).
Fig. 3. Images of tadpoles and frogs derived from oocytes and embryos injected with Tyr-TALEN mRNAs.
(A) Wild-type stage 39–40 tadpoles. (B,C) Fertilized eggs were injected with 500 pg of Tyr-TALEN mRNAs. Four of the stage 39–40 tadpoles were derived from the eggs from two different female frogs (B) and (C). (D) Oocytes were injected with Tyr-TALEN mRNAs, transferred into a host female, and fertilized. Each pair of stage 39–40 tadpoles is colored green, brown, mauve, or red from left to right. The green- and brown-colored tadpoles were derived from oocytes injected with 250 pg of mRNA. The mauve- and red-colored tadpoles were derived from oocytes injected with 500 pg of mRNA. The oocytes were matured by progesterone at 18°C for 11 hours for the green- and mauve-colored tadpoles. The oocytes were matured by progesterone at 16°C for 16 hours for the brown- and red-colored tadpoles. There was no difference between oocytes treated with progesterone at 18°C for 11 hours and those treated at 16°C for 16 hours. Tadpoles were selected randomly. (E) An image of the frogs derived from the oocytes that were injected with Tyr-TALEN mRNAs. Five of seven frogs showed perfect albino phenotypes. Scale bars = 1 mm (A–D) or 1 cm (E).
Fig. 4. Time course of TALEN-mCherry fusion protein expression in oocytes and embryos.
(A,D) Schematic representation of the Tyr-TALEN-mCherry (A) and Tyr-TALEN-mCherry-DS (D) mRNAs. (B,E) Oocytes were injected with either Tyr-TALEN-mCherry (B) or Tyr-TALEN-mCherry-DS (E) mRNAs, transferred into a host female, and fertilized. Five oocytes or host-transferred embryos were homogenized at the indicated time and examined by Western blot analysis using anti-DsRed and anti-actin antibodies. The closed and open arrowheads indicate the positions of the Tyr-TALEN-mCherry and actin proteins, respectively. The stage 13 embryos in the right-most lane were not injected with mRNAs. (C,F) Quantification of the Tyr-TALEN-mCherry protein signals shown in (B,E). The Tyr-TALEN-mCherry proteins were normalized to actin and the signal for the lysates of the stage 13-injected embryos was set to 100%. P, progesterone-treatment.
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