Fig. 7
Generation of an allelic series of mutations at the Grhl3 NLS knock-in locus. a Strategy for the production of an allelic series of mutations at the Grhl3 nuclear localization signal (NLS) mutant locus. A schematic representation of the Grhl3 wild-type allele, targeting vector, Grhl3NLS-neo and Grhl3NLS alleles. b Identification and characterization of mutant alleles by southern blot analysis. Genomic DNA samples from wild-type (Wt) and mutant mice were digested with NcoI and hybridized with the DNA probe (from +551 to +1194) to examine the correct 3′ recombination and excised pgk-neo cassette. The 3.9-kb and 2.5-kb bands represent wild-type (Wt) and targeted NLS-neo alleles. The mutant 2.5-kb band shifted up to 3.9 kb after Flpe excision in the NLS allele. c The Grhl3NLS allele produced Grhl3 transcripts from the inserted site of NLS signal. Reverse transcription (RT)–PCR analysis with cDNA from wild-type, Grhl3ΔEx2/ΔEx2 and Grhl3NLS/NLS mutant embryos at E11.5. The 171-bp products correspond to wild-type transcripts, whereas the 192-bp products correspond to mutant transcripts that contain an additional NLS signal sequence. d–f Whole mount in situ hybridization analysis of Grhl3 transcripts in wild-type (d), Grhl3ΔEx2/ΔEx2 (e), and Grhl3NLS/NLS (f) mutant embryos at E8.0. Grhl3 transcripts were clearly detected at the boundary between surface ectoderm and neuroectoderm in wild-type (d, arrows) and Grhl3NLS/NLS mutant (f, arrows) embryos while they were undetected in the Grhl3cre/cre embryo (e). g Western blots using the GRHL3 antibody aa195–211 (Supplementary Fig. 3g, 5c) with protein extracts of embryonic nucleus. Bands of around 67 kDa, corresponding to full-length GRHL3 products, were evident in wild-type and NLS alleles but not Δex2 alleles. h–k Identification of nuclear-localized GRHL3 protein in Grhl3NLS/NLS mutant embryos. Immunohistochemistry of GRHL3 (anti-GRHL3 ab; aa478–aa493; Supplementary Fig. 5e; green) and DAPI (magenta). GRHL3 protein was mostly distributed in the nuclei of SE cells of a Grhl3NLS/NLS embryo (j, k), while it localized to the cytoplasm mainly and partly nucleus in SE cells of a wild-type embryo at E8.5 (h, i). Representative images from more than two independentexperiments. Scale bars represent 50 (h, j) and 200 μm (d–f)
