Inactivation of the CAP2/<i>Tmprss4</i> gene locus.

<p><b>(A)</b> Scheme of the wild-type allele, the targeting vector, and the targeted CAP2/<i>Tmprss4</i>^{<i>loxneo</i>} allele following homologous recombination, and the CAP2/<i>Tmprss4</i>^<i>lox</i> and the CAP2/<i>Tmprss4</i>^Δ allele following breeding with Flp- and Cre-deleter mice, respectively. Relevant restriction enzymes for cloning and diagnosis of targeted ES cell clones are shown. Exons 8 and 9 and the neomycin cassette (flanked by <i>frt</i> sites) are flanked by <i>lox</i>P sites. 5’ and 3’ probes as well as PCR primers used for ES cell screening and mouse genotyping are indicated. (<b>B</b>) Southern blot analyses of targeted ES cell clones using the external 5’probe (upper left panel) following digestion with <i>Spe</i>I and <i>Nhe</i>I, the neo probe (upper right panel) following <i>Eco</i>RI digestion, and the external 3’probe following digestion with <i>Bam</i>H1; note that clone #2 and #3 harbour additional recombination and integration events as evidenced by Southern blot analyses using the 5’ and neo probe, respectively. (<b>C</b>) Southern blot analysis of CAP2/<i>Tmprss4</i>^{<i>loxneo/+</i>}, CAP2/<i>Tmprss4</i>^{<i>lox/lox</i>} and/or CAP2/<i>Tmprss4</i>^<i>lox/+</i> and CAP2/<i>Tmprss</i>^Δ<i>/</i>Δ mice using the 5’ probe following <i>Spe</i>I/<i>Nhe</i>I digestion. (<b>D</b>) PCR-based genotyping of mice harbouring the wild type (<i>+</i>, 250bp, lane 1 and 3), <i>lox</i> alleles (<i>lox</i>, 350bp, lane 2) and knockout alleles (Δ, 500bp, lane 3 and 4).</p

ENaC mRNA transcript and protein expression in kidneys from CAP2/<i>Tmprss4</i> wildtype (WT), heterozygous mutant (HET) and knockout (KO) mice under sodium-deficient diet.

<p><b>(A-C)</b> Relative mRNA transcript and (<b>D-F</b>) protein expression of (<b>A</b>) <i>Scnn1a</i>, (<b>B</b>) <i>Scnn1b</i> and (<b>C)</b><i>Scnn1g</i> from CAP2/<i>Tmprss4</i> wildtype (WT), heterozygous mutant (HET), and knockout (KO) mice; n = 4 for each group and genotype; β-actin was used as internal control. Representative immunoblots of (<b>D)</b> Scnn1a, (<b>E</b>) Scnn1b and (<b>F</b>) Scnn1g and its corresponding β-actin protein expression from CAP2/<i>Tmprss4</i> wildtype (WT), heterozygous mutant (HET) and knockout (KO) mice (n = 5 for each group and genotype); kidney extracts from Scnn1 wildtype (WT) and knockout (KO) mice were used as positive and negative control respectively; arrows indicate the full-length and the corresponding cleaved ENaC fragments; * <i>P</i>< 0.05).</p

ENaC mRNA transcript expression and activity in colon from CAP2/<i>Tmprss4</i> mice under sodium-deficient diet.

<p>(<b>A-C</b>) Relative mRNA transcript expression of (<b>A</b>) <i>Scnn1a</i>, (<b>B</b>) <i>Scnn1b</i> and (<b>C)</b><i>Scnn1g</i> from CAP2/<i>Tmprss4</i> wildtype (WT, n = 4), heterozygous mutant (HET, n = 5), and knockout (KO, n = 4) mice; *<i>P</i>< 0.05); β-actin was used as internal control. (<b>D)</b> Morning and afternoon amiloride-sensitive rectal potential difference (PD) measurements at 10-12am and 4-6pm of two consecutive days in <i>Tmprss4</i> wildtype (WT), heterozygous mutant (HET) and knockout (KO) mice; n = 4 for each group and genotype.</p

Histopathological analysis in ENaC-expressing organs from CAP2/<i>Tmprss4</i> knockout mice.

<p>Representative H&E stained section of colon, lung, kidney and skin from CAP2/<i>Tmprss4</i> wildtype (WT), heterozygous mutant (HET) and knockout (KO) mice; n = 2 females and 2 males for each group and genotype; bar indicates 100μm.</p

Phenotype of CAP2/<i>Tmprss4</i>-deficient mice.

<p><b>(A)</b> Representative pictures of 3 months old (male) CAP2/<i>Tmprss4</i> wildtype (WT) and CAP2/<i>Tmprss4</i> knockout (KO) littermates. (<b>B</b>) Mean body weight (g) of 3-month-old male and female wildtype (WT, n = 6), heterozygous mutant (HET, n = 11 and n = 9, respectively), and knockout (KO, n = 6 and n = 5, respectively) mice. (<b>C</b>) Relative CAP2/<i>Tmprss4</i> mRNA transcript expression in colon from CAP2/<i>Tmprss4</i>^<i>WT</i>, CAP2/<i>Tmprss4</i>^<i>HET</i> and CAP2/<i>Tmprss4</i>^<i>KO</i> mice (n = 6 mice per group); β-actin is used as internal control. (<b>D</b>) Representative immunoblot showing the presence of a 70kDa CAP2/Tmprss4-specific band in colon extracts from CAP2/<i>Tmprss4</i>^<i>WT</i> (lane 1 and 2), CAP2/<i>Tmprss4</i>^HET (lane 3–5) mice and absence in CAP2/<i>Tmprss4</i>^<i>KO</i> (lane 6–8) mice; arrow indicates the size of the expected but absent CAP2/Tmprss4-specific band in knockouts.</p

Distribution of wildtype CAP2/<i>Tmprss4</i> mRNA transcript expression.

<p>CAP2/<i>Tmprss4</i> mRNA expression profile in WT mice (n = 4) in various organs as indicated; individual values were normalized to β-actin.</p