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A gene of interest can be efficiently modified using transcription activator-like effector nucleases (TALENs) (Christian et al., 2010;Li et al., 2011). However, if a target gene is essential for development, growth and fertility, use of TALENs with high mutagenic activity in F0 frogs could result in developmental disorders or sterility, which would reduce the number of F1 progeny and make F1 phenotypical analysis difficult. We used the 3' untranslated region of DEADSouth gene (DS-3') of Xenopus tropicalis to solve this problem, because the addition of the DS-3' to mRNA is known to induce primordial germ cell (PGC)-specific expression and reduce the stability in somatic cells of mRNA in Xenopus laevis. At first, we inserted the X. tropicalis DS-3' downstream of the EGFP termination codon and confirmed that the EGFP expression was specifically detected in PGCs for three weeks. Therefore, we inserted the DS-3' downstream of the termination codon of the TALEN coding sequence. The tyrosinase gene was selected as the target gene for TALEN because the bi-allelic mutation of this gene is easily discernible by the albino phenotype. When fertilized eggs were microinjected with TALEN mRNAs fused to the DS-3', their sperm and oocytes had a high rate (84-100%) of target-gene modification in contrast to the lower rate (0-45%) of nucleotide alteration observed in somatic cells.
Fig. 1. Expression of EGFP in X. tropicalis tadpoles injected with EGFP-DS mRNA.
(A,H) A schematic representation of a lateral view of an 8-day-old tadpole (A) and a ventral view of a 21-day-old tadpole (H). (B–G) Higher-magnification view of A. (I–N) Higher-magnification view of H. (B,E,I,L) Images of wild-type tadpoles. (C,F,J,M) Images of tadpoles injected with EGFP mRNA. (D,G,K,N) Images of tadpoles injected with EGFP-DS mRNA. (B–D) Brightfield images of live 8-day-old tadpoles. (E–G) EGFP expression in B, C and D, respectively. (I–K) Brightfield images of the mesonephros of 21-day-old tadpoles. (L–N) EGFP expression in I,J and K, respectively. F and G show the ratio of the number of EGFP-positive tadpoles on the eighth day to the number of mRNA-injected tadpoles. M and N show the ratio of the number of EGFP-positive tadpoles on the 21st day to the number of EGFP-positive tadpoles on the eighth day. The white arrowheads indicate PGCs. Scale bars = 1 mm in (E–G) and 0.1 mm in (L–N).
Fig. 2. Phenotype of F0 frogs.
(A) Photographs of F0 frogs. Tyr-TALEN-DS mRNAs were co-injected with EGFP-DS mRNA at the vegetal pole of fertilized eggs. The frogs were allowed to reach sexual maturity. Scale barsâ=â1â cm. (B) Ovary of a wild-type one-year-old frog. (C) Ovary of a one-year-old F0 frog that was injected with Tyr-TALEN-DS mRNAs. (B,C) Scale barsâ=â1â mm.
Fig. 3. Analysis of individual offspring obtained from crosses between F0 and albino frogs.
(A) Schematic representation showing both the gene mutations of the albino mates used in the crosses and the locations of primers used in the mutation analysis. The bold horizontal line indicates the DNA region from the initiation codon to the end of the first exon. The mutated sequences of the albino mates are shown as type I, type II and type III with the wild-type sequence. The inserted and deleted nucleotides are indicated with a red character and blue dashes, respectively. The locations of the mutations and the 365-bp deletion in the albino mates are indicated by a black arrow and rectangle, respectively. The Tyr-TALEN target site and the direction of the primers are indicated by a purple arrow and blue arrows, respectively. (BâD) Brightfield images of live wild-type (B), lightly pigmented (C) and albino (D) tadpoles. Note that the abdominal region is lightly pigmented in C. Scale barsâ=â1 mm. (E) Phenotypic analysis of the offspring of F0 and albino frogs. The percentages of wild-type, lightly pigmented and albino F1 tadpoles are indicated by black, gray and white bars, respectively (Tableâ 1). The numbers in the bars are the percentages of tadpoles with the indicated phenotype. The total number of tadpoles is shown at the top of the bars. (F) Genotypic analysis of individual offspring of F0 and albino frogs. The target DNA fragment was amplified using genomic DNA purified from individual wild-type, lightly pigmented and albino F1 tadpoles and was recloned for sequence determination. All of the mating data are shown. Different sequences were bequeathed by F0 frogs to different F1 offspring, which also had the other mutated tyrosinase allele inherited from the albino parent. The target sequences derived from the albino mates are not shown. The wild-type target DNA and the amino acid sequences are indicated at the top and bottom of the panel, respectively. A pair of purple bars denotes the TALEN-binding sites. The gaps resulting from a deletion (Î), the inserted nucleotides (+) and the exchanged nucleotides (*) are indicated with blue dashes, red characters and green characters, respectively. The mutation types are indicated on the right. The ratio of the number of tadpoles with the indicated sequence to the total number of tadpoles with the same phenotype is shown in parentheses on the right. Large deletions of 620 and 952 nucleotides were observed in the offspring derived from the f1 and f2 frogs, respectively, and both of these deletions contained the start codon.
Fig. 4. Analysis of individual offspring of F0 frogs.
(A) Phenotypic analysis of the offspring obtained by mating two F0 frogs, m1 and f1. The percentages of wild-type and albino F1 tadpoles are indicated by black and white bars, respectively, and the values are denoted in the bars. The total number of tadpoles is shown on the right. (B) Genotypic analysis of individual offspring obtained from mating the m1 and f1 frogs. The target DNA fragment was amplified using genomic DNA that was purified from individual F1 tadpoles and was recloned for sequence determination. The phenotype (Ph) and genotype (Ge) of F1 tadpoles are indicated on the left. Each F1 tadpole had a pair of sequence lines as shown in brackets. wt, wild-type target sequence; in, in-frame mutation; out, out-of-frame mutation; L, large deletion containing the start codon or the exon-intron boundary. The ratio of the number of tadpoles with a pair of the indicated sequences to the total number of tadpoles with the same phenotype and genotype is shown in parentheses on the right. The alignment is labeled as described in the legend of Fig. 3F.
Fig. 5. Preferential mutagenesis in the testes of the m2 frog.
(A) Simple direct sequencing of the PCR-amplified targeted genome region (DSP assay) using genomic DNA obtained from several organs and tissues of the Tyr-TALEN-DS-mRNA-injected m2 frog. The spacer sequence located between the Tyr-TALEN binding sites is shown at the top of the panel. The ambiguous sequence in the right and left testes begins with âAâ (a red wave) and extends to the right, as shown by the red arrows, suggesting that these amplicons are mixtures of heterogeneous fragments with different mutations. (B) Mutated target sequences in organs and tissues of the m2 frog. The target DNA fragment was amplified using genomic DNA that was purified from organs and tissues and subcloned. The sequences of eight to 12 clones per organ or tissue were determined. The ratio of the number of the indicated sequence to the total number of sequences in an organ or tissue is shown in parentheses on the right. The alignment is labeled as described in the legend of Fig.â 3F. (C) Mutation hit rate in several organs and tissues of m2. The mutation hit rate in an organ or tissue is indicated by a black bar, whereas the estimated mutation rate in the m2 spermatozoa is indicated by a white bar.
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