TALEN design and evaluation of cutting efficiency in rat glioma C6 cells.

<p>(a) Schematic of the rat <i>Nc3r1</i> (GR) gene. Zoom on the area of the mutation pA476T in exon 3. The first nucleotide of the 476 codon is highlighted in blue. TALEN binding sites of TAL 3 are highlighted in green. Detailed sequences of TAL 6 binding sites can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088146#pone.0088146.s001" target="_blank">Table S1, File S1</a>. (<b>b</b>) T-endo1 assay results. Pooled DNA from C6 cells transfected with either Right, Left or Right and Left TALEN monomers (marked with R, L or RL, respectively) was amplified and treated with T7 endo 1 enzyme. Cut bands of 288 and 177 bp indicate TALEN activity. Mcells are mock transfected cells, GFP: GFP transfected cells were used as a positive transfection control. Intensity of the cut bands are indicated for TAL 3 and TAL 6 pairs. (<b>c</b>) TAL 3 transfected cells screening. PCR amplicons of the region around the pA76T mutation were subcloned into TOPO vector. Clones were isolated and analyzed individualy. Four point mutations and insertion.s are marked in red.</p

Fo KO rat genotyping.

<p>Wt, wild type sequence. TALEN binding sites are shown in green. The pA476T mutation is highlighted in blue. Longer deletions are marked with double slash. Rats 11.4, 6.1 and 5.5 (underlined) were kept for breeding. All rats beared the wt allele of the <i>Nr3c1</i> gene. Primers used are listed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088146#pone.0088146.s001" target="_blank">Table S2, File S1</a></b>.</p

Injections of TAL 3 mRNA and donor plasmid DNA in rat one-cell embryos.

†<p>One pup was born dead.</p><p>Two doses of TAL 3 mRNA were used (20+20 or 10+10 ng/µl of each TALEN). The egg survival rate is shown in percentage. NHEJ indicates the number of pups that had a gene disruption event in the sequence around pA476T. The percentages were calculated within each set of TALEN mRNA amount injected.</p

Founder KI female 3.4 genotyping from subcloned PCR amplicons of tail biopsies.

<p>Wt: Wild type; DP: donor plasmid. Point mutations in the DP are indicated in red bold letters. The pA476T mutation is highlighted in blue.</p

Detection of random donor integration in rat founders.

<p>Representative Southern blot analysis of founder rat genomic DNA following <i>Hin</i>cII digestion of genomic DNA and hybridization with exon 3-derived probe. Indicated is the 3.6 kb endogenous exon 3-containing genomic fragment and additional random integrations in founder 5.4 (knockout) and 8.1 (wildtype) rats. Rats 5.5 and 6.1 are knockouts as well but presented no off-target donor integration.</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

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 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