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Figure S5. Generation and characterization of the MALS-3 floxed allele. (A) Restriction map of the native murine MALS-3 gene (top), the pks2loxPFRTNT targeting plasmid (middle, top), and the targeted locus before (middle, bottom) and after (bottom; ΔNeo) removal of the neomycin cassette. Filled black bars indicate MALS-3 exons; green arrowheads indicate loxP sites; orange arrowheads indicate FRT sites; red filled bars denote Southern probes; and blue filled bars specify PCR genotyping fragments. The following abbreviations are used: B, BamHI; E, EcoRI; H, HindIII; Neo, neomycin. (B) Southern blots of BamHI digested genomic DNA from wild-type (+/+) and heterozygote for the floxed allele (+/flox). The 5′ probe anneals to a single ∼19-kb band in the lane containing +/+ DNA and an additional 8.6-kb band is present in the lane containing genomic DNA from +/flox. Additionally, the neo probe anneals to a band at the predicted 3.9-kb size in a lane containing +/flox genomic DNA and the 3′ probe anneals to a fragment at ∼19 kb in the +/+ lane and an additional 8.3-kb band in the lane containing DNA from +/flox. (C) PCR genotyping of +/+, +/flox(ΔNeo), and flox/flox(ΔNeo) mice yields 370- and 445-bp products for the wild-type and targeted loci, respectively. (D) Cre-dependent expression of EYFP (green) in E12 Pax3-Pro embryos is restricted to the metanephric mesenchyme and absent in the ureteric bud (red, cytokeratin). (E) Immunohistochemistry of dissected ureteric buds from E11.5 MALS-3 flox/flox embryos expressing cre under the control of Pax3 (MMKO) or HoxB7 (UBKO) promoters. MALS-3 (red) expression is specifically missing in the ureteric buds from UBKO (bottom) mice but not from MMKO (top) mice. Cytokeratin (green) was used as a ureteric bud marker. (F) Immunohistochemistry of kidney sections from MALS-3 flox/flox control (−CRE) mice or MALS-3 flox/flox mice expressing cre under the control of either HoxB7-Pro (UBKO) or Pax3-Pro (MMKO). Antibody against AQP2 (green) was used to label collecting ducts (asterisks). In control mice (top), MALS-3 (red) localizes to the basolateral membrane of most tubules. UBKO mice (middle) specifically lack expression of MALS-3 (red) in the collecting ducts (asterisks), whereas MALS-3 expression is unchanged in other tubules. In contrast, MALS-3 (red) properly localizes at the basolateral membrane of most collecting ducts (asterisks) in MMKO mice (bottom), but tubules derived from the metanephric mesenchyme lack MALS-3 expression. Bar = 50 μm in D and E, and 10 μm in F.