Figure 1: Generation of Sox9-3′ EGFP knock-in mice and Sox9-EGFP/EGFP null chimeras.
From: Wwp2 is essential for palatogenesis mediated by the interaction between Sox9 and mediator subunit 25
(a) The structure of the genomic Sox9 locus, targeting vector and targeted allele for Sox9-3′ EGFP knock-in mouse is indicated. Exons are depicted as closed boxes and intronic sequences are shown as solid lines. DNA fragments detected by Southern blot analysis are indicated by double arrows, with the restriction enzymes and the probe. The IRES-EGFP-pA/loxP-flanked PGK-neo-bpA cassette is depicted as open boxes. B, BamHI; P, Pst1; E, EcoRI; X, XbaI; Bg, BglII; H, HpaI. (b) Southern blot analysis of genomic DNA. Genomic DNA isolated from ES cell clones was digested with BglII and was then hybridized with the 3′ probe. The wild-type and mutant alleles were detected as 3 kb and 6.5 kb fragments, respectively. (c) EGFP expression in E9.5, E13.5 and E16.5 Sox9-3′ EGFP knock-in embryos. EGFP expression was detectable in a Sox9-specific pattern during embryogenesis. Scale bars, 500 μm (E9.5); 2 mm (E13.5) and 3 mm (E16.5). (d) The structure of the genomic Sox9 locus, targeting vector and targeted allele for Sox9-EGFP/EGFP null chimera is indicated. DNA fragments detected by Southern blot analysis are indicated by double arrows. (e) Southern blot analysis of genomic DNA. Genomic DNA isolated from ES cell clones was digested with EcoRI and was then hybridized with the 3′ probe. Wild-type and mutant alleles were detected as 7.1 kb and 5.8 kb fragments, respectively. (f) Southern blot analysis of the neo-deleted Sox9 EGFP clones after Cre recombination. The neo-deleted Sox9 EGFP allele was detected as a 4.2 kb fragment. (g) Southern blot analysis of the neo-deleted Sox9 EGFP/EGFP-neo clones. (h) Dot plot cytograms show enrichment of EGFP-positive cells in Sox9-3′ EGFP knock-in embryos (top panel) and Sox9-EGFP/EGFP null chimeras (bottom panel). Only cells from a P1 population showing high green fluorescence were sorted. (i) RT–PCR analysis of Sox9 expression using total RNA extracted from sorted EGFP-positive cells of Sox9-3′ EGFP knock-in embryos and Sox9-EGFP/EGFP null chimeras.
