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Completion of the Xenopus laevis genome sequence from inbred J strain animals has facilitated the generation of germline mutant X. laevis using targeted genome editing. In the last few years, numerous reports have demonstrated that TALENs are able to induce mutations in F0 Xenopus embryos, but none has demonstrated germline transmission of such mutations in X. laevis. In this report we used the oocyte host-transfer method to generate mutations in both tyrosinase homeologs and found highly-penetrant germline mutations; in contrast, embryonic injections yielded few germline mutations. We also compared the distribution of mutations in several F0 somatic tissues and germ cells and found that the majority of mutations in each tissue were different. These results establish that X. laevis J strain animals are very useful for generating germline mutations and that the oocyte host-transfer method is an efficient technique for generating mutations in both homeologs.
Fig. 1. Examples of F0 and F1 X. laevis tyr mutants. (A) Schematic diagram of tyrosinase locus, showing the five exons. The scissors show where the TALENs were designed to target the 3′ end of the first exon, and the arrows show the location of primers used for genotyping. (B) Picture of an 8-month-old tyr mutant F0 J strain frog generated by oocyte host transfer. Notice small patch of pigmentation in the body and eye (arrows). (C) Picture of an 8-month-old F0 tyr mutant frog generated by injection of TALEN mRNAs into one-cell stage embryos. (D) Picture of natural mating of two tyr mutant F0 frogs (OHT female and OHT male #1) at 14 months. (E) Picture of an F1 tyr mutant frog from mating of OHT female and OHT male #1 at 6 months of age.
Fig. 2. Genotyping of F1 tyr mutant embryos. (A) Bar graph illustrating the frequency of tyr.L and tyr.S mutations in F1 embryos generated by mating F0 OHT frog with wild type J strain. Wild type alleles make up the remainder of each column. (B) Sequence of mutations identified in F1 embryos. Frequency refers to the number of F1 embryos that contained the mutation. Red font indicates TALEN target sites.
Fig. 3. Genotyping of F0 somatic tissue of female OHT tyr mutant frog. (A) Sequence of tyr.L mutations identified in the heart, liver, skin, gut and lung of F0 female OHT frog. (B) Sequence of tyr.S mutations identified in heart, liver, skin, gut and lung of F0 female OHT frog. Frequency refers to the number of clones that contained the mutation. Red font indicates TALEN target sites.
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