Fig 2: Presence of proteins in the Ets2 mutant skin and thymus. Western blotting with anti-Ets2 antibody and anti-Gapdh antibody in the skin (A) and thymus (B). Native Ets2 protein (*) was detected only in WT samples. Ets2 ?Ex8 (**) and Ets2 em1 (***) mutant proteins were not detected in WT and null mutant samples. Ets2 em2 mutant protein (the predicted size indicates as ##) were not detected in Ets2em2/em2 (em2) skin.

Fig 3: Development of Ets2em1/em1 embryos. (A) From E7.5 to E10.5, embryos were observed after crossing Ets2+/em1 females and males. Ets2em1/em1 embryos are indicated as circles in each picture. The regions outside of the circles correspond to Ets2+/+ or Ets2+/em1 embryos. The genotype of the embryo indicated by an asterisk at E9.5 could not be determined. All scale bars indicate 1 mm. (B) Gene expressions of E7.5 WT and Ets2em1/em1 (em1) embryos. Embryos were separated into trophectodermal tissue (TE), including ectoplacental corn, and epiblast (Epi), from which RNA and cDNA were prepared. Both em1 bands were shifted to be lower than the WT bands. The DNA sequences of the em1 bands were the same as em1L in Fig. 4B.

Fig 4: Gene expression in the Ets2em1/em1 skin. (A) Gene expression from the Ets2 locus in skins of WT, Ets2null(em3/em3) mutant (null) and Ets2em1/em1 (em1) mice. From em1 skin, two types of mRNA were expressed, although both were shorter than WT. The larger mRNA was called em1L, and the smaller mRNA was called em1S. (B) Sequences of em1L and em1S. Em1L was the expected sequence, but em1S contained a deleted locus that matched exon 8, shown in Supplemental Fig. S5. (C) Gene expression of Mmp3 and Mmp9 in the Ets2em1/em1 skin. There were significant differences in the expression level of Mmp3 between WT and null, em1 and null, and WT and em1. The expression level of Mmp9 in the null skin was slightly decreased compared with WT, but not significantly, and em1 showed a significant difference compared with null. *p < 0.05, **p < 0.01.

Fig 5: Generation of new Ets2 mutant models using zygotes. (A) Design of crRNA targeting sites in exon 8 of the Ets2 gene and checking primer positions. (B) Strategy for obtaining Ets2 homozygous mutant l in F0 generation using the electroporation technique and the tetraploid method. (C) Genotyping of the two newly generated Ets2 mutants in F0 generation. Both had a wildtype (WT: 564 bp) allele and a deletion allele (em1 or em2), which were detected as shorter bands than WT. (D) DNA sequence of mutant alleles and crRNA targeted sequences. The 205 bp deleted (?205 bp) allele was named em1, and the 82 bp deleted (?82 bp) allele was named em2.