Persistent expression of Pax3 in the neural crest causes cleft palate and defective osteogenesis in mice
J. Clin. Invest. Meilin Wu, et al. 118:2076 doi:10.1172/JCI33715 [Go to this article.]

Figure 1
Pax3 knockin at Rosa26 locus. (A) The murine Pax3 cDNA was inserted downstream of a PGK-neo cassette with a transcriptional stop signal flanked by 2 loxP sites in the pBigT plasmid containing the Rosa26 genomic sequence. Homologous recombination between the Rosa26Pax3 construct and the appropriate genomic locus resulted in a targeted allele where Pax3 is expressed only after Cre-mediated recombination. A probe for Southern blot confirmation of homologous recombination is indicated. (B) Southern blot analysis of WT and R26^Pax3/+ EcoRV-digested genomic DNA from wild-type and targeted ES cells showing wild-type and knockin (KI) bands. (C) Western blot of lysates from uninfected (lane 1) mouse embryonic stem cells targeted with R26^Pax3/+ or infected with Cre-adenovirus (lane 2) with Pax3 antibody. Pax3 protein from transfected 293T cells was used as a positive control (lane 3). (D) Cre-adenovirus–infected R26^Pax3/+ ES cells immunostained with Pax3 antibody. Original magnification, ×100. (E) Immunohistochemistry to detect Pax3 protein in the smooth muscle layer of the aortic arch (arrows) of E16.5 embryos. Pax3 protein is detected in R26^Pax3/Pax3 embryos only when Pax3Cre (right panel) is present. Endothelial and luminal staining is artifactual. Pax3 staining is nuclear. Original magnification, ×25. (F) Western blot to detect Pax3 protein in E11.5 whole-embryo lysates. Actin is shown as control, and relative pixel density is indicated below each lane.