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

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

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

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

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

Renal Ca2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5.

Contains fulltext : 29917.pdf (publisher's version ) (Open Access

Lack of Renal Tubular Glucocorticoid Receptor Decreases the Thiazide-Sensitive Na+/Cl– Cotransporter NCC and Transiently Affects Sodium Handling

International audienceChronic glucocorticoid infusion impairs NCC activity and induces a non-dipping profile in mice, suggesting that glucocorticoids are essential for daily blood pressure variations. In this paper, we studied mice lacking the renal tubular glucocorticoid receptor (GR) in adulthood (GR knockouts, Nr3c1Pax8/LC1). Upon standard salt diet, Nr3c1Pax8/LC1 mice grow normally, but show reduced NCC activity despite normal plasma aldosterone levels. Following diet switch to low sodium, Nr3c1Pax8/LC1 mice exhibit a transient but significant reduction in the activity of NCC and expression of NHE3 and NKCC2 accompanied by significant increased Spak activity. This is followed by transiently increased urinary sodium excretion and higher plasma aldosterone concentrations. Plasma corticosterone levels and 11βHSD2 mRNA expression and activity in the whole kidney remain unchanged. High salt diet does not affect whole body Na+ and/or K+ balance and NCC activity is not reduced, but leads to a significant increase in diastolic blood pressure dipping in Nr3c1Pax8/LC1 mice. When high sodium treatment is followed by 48 h of darkness, NCC abundance is reduced in knockout mice although activity is not different. Our data show that upon Na+ restriction renal tubular GR-deficiency transiently affects Na+ handling and transport pathways. Overall, upon standard, low Na+ and high Na+ diet exposure Na+ and K+ balance is maintained as evidenced by normal plasma and urinary Na+ and K+ and aldosterone concentrations

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