Impact of inducible Arg1 knockout on phenotypic presentation.

<p>(A) Typical appearance of Arg1 knockout mouse at humane endpoint (right), with healthy-appearing vehicle-treated control at the same timepoint (left). (B) Percent changes in body weight during the experimental period relative to body weights taken four days following injections are shown on y-axis. (C) Kaplan-Meier survival curve comparison depicts that tamoxifen-treated mice display significantly reduced survival rates when compared to ROSA and vehicle-treated mice. Data are mean ± SEM for n = 5–11 in each group. Statistical significance between groups was determined by Student's <i>t</i>-test (*<i>P<0.05</i>). (D) Quantitative analysis of walking footprint patterns based on measurements of stride length, forepaw base and hindpaw base width, and distance between front and hind footprint placement. (n = 6).</p

Down-regulation of Arg 1 expression in tamoxifen-treated Arg-Cre mice.

<p>(A) Transcriptional response of <i>Arg1</i> to tamoxifen induction. Real-time qPCR analysis of <i>Arg 1</i> gene expression was performed in liver, brain and kidney tissues 12 days after tamoxifen administration. Differences in RNA input for reverse transcription were normalized using Ct values obtained in parallel for mouse 18S rRNA. Fold change was calculated relative to the vehicle-treated samples using the comparative threshold method (2^−ΔΔCt). Values are mean ± SEM for n = 3–6 in each group. Statistical significance between groups was determined by Student's <i>t</i>-test. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001. (B) Western blot analysis of total liver extracts (20 µg/well) from four age groups. Arg1 protein expression was evaluated by immunoblotting with an anti-Arg1 antibody (C-terminal) to confirm the level of knockdown. α-tubulin was used as loading control. Representative immunoblot from three independent experiments is shown. (C) Real-time qPCR analysis of <i>Arg 2</i> gene expression in kidney.</p

Generation of inducible Arg1-deficient mice.

<p>(A) Experimental setup for the gene targeting strategy. <i>Arg1^flox</i> mice were crossbred with CreER^T2 mice to generate Arg1-Cre mice. Four separate groups of mice have been tested for tamoxifen-mediated Cre removal of exons 7 and 8 of <i>Arg1</i>. The arrows depict the locations of primers used for genotyping the resulting mice with approximate sizes of the different PCR products shown. (B) Representative agarose gel of PCR genotyping using genomic DNA from ear punch or tail biopsies of vehicle- and tamoxifen-treated Arg-Cre mice. Arg1-Cre mice exhibited two bands at 1.2 kb and 252 bp, while knockout mice showed a single band at 195 bp.</p

Tamoxifen-mediated inducible Arg1 knockout leads to hyperammonemic crisis.

<p>Plasma ammonia assay performed at five different time-points of three age groups. Values are expressed as percentage change in plasma ammonia concentration in tamoxifen-treated mice compared to vehicle-treated control mice. Ammonia concentrations ranged between 305–595 µmol/L (vehicle-treated mice) and 903–1791 µmol/L (tamoxifen-treated mice). Data are mean ± SEM for n = 3–5 in each group. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001.</p

Plasma amino acid analysis of mice from different age groups (pooled) measured at humane endpoint.

<p>Values are mean ± SEM for n = 11–12 and are expressed as micromoles per liter (µmol/L). BCAA, branched chain amino acid; NS, not significant.</p

Reduced arginase enzyme activity and urea production in tamoxifen-treated Arg-Cre mice.

<p>(A) Arginase activity in liver tissue extracts from vehicle- and tamoxifen-treated mice. The livers were homogenized and the arginase enzyme activity assay was carried out as mentioned in the “<i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080001#s2" target="_blank">Materials and Methods</a></i>” section. (B) Functional capacity of hepatocytes assessed by determining urea production. Isolation of primary mouse hepatocytes was performed based on the two-step collagenase perfusion technique. Typical morphology of mouse hepatocytes cultured on a single layer of collagen gel at 24 h (top panel). Hepatocyte urea production was spectrophotometrically determined at 544 nm (bottom panel). Values are mean ± SEM for n = 3–7 in each group. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001.</p

Some male mice are rescued from the lethal consequences of induced Arg1 deletion by AAVrh10.CB7.CI.Arg1-eGFP.WPRE.rBG administration.

<p>(A) Survival of tamoxifen-treated male (n = 24) and female (n = 10) induced Arg1 KO mice injected with AAV vector (Expts 4–7, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.t001" target="_blank">Table 1</a>). Seven “rescued” healthy mice were sacrificed at Day +21 (mouse A2, n = 1), Day +49 (mice S9, S11; n = 2), Day +90 (mice S1, S4; n = 2), and Day +180 (mice A1, A10; n = 2) for analysis of Arg1-eGFP expression. This explains the drop off in male survival beyond Day +21 denoted by asterisks. (B) <i>In situ</i> liver showing extensive green fluorescence expression from the Arg1-eGFP transgene in one rescued male mouse S1 sacrificed at Day +90, viewed under a handheld UV light (purple background). Note absence of expression in the gall bladder. (C) Western blot depicting Arg1-eGFP transgene expression in six of the rescued male mice mentioned above (mouse A2 not tested). The lane corresponding to mouse S6 was non-AAV-treated (Arg activity = 0.24 units; not shown in panel D). Protein standards (std) are not visible on the Western blot. Con, control non-tamoxifen treated mouse injected with an AAV-GFP construct. (D) Liver arginase enzyme activity at endpoint in AAV-injected mice of Expts 4–7 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.t001" target="_blank">Table 1</a>). Activity of rescued (A1, A2, A10, S1, S4, S9, S11) mice are labeled, along with three mice not rescued but with substantial enzyme activity (S8, S10, S12).</p

Transgenic expression of Arg1-eGFP rescues some male Arg1 knockout mice but variably corrects plasma arginine.

<p>(A) Phase contrast cryostat section (non-fixed, non-stained; left panel) and corresponding fluorescent image of liver section from mouse S9 (right panel) analyzed on Day +49 administered a single dose of 7.5x10^<i>10</i> gc AAVrh10.CB7.CI.Arg1-eGFP.WPRE.rBG 68 days earlier. Note widespread expression beyond boundaries of vessels. (B, C) Plasma arginine concentrations expressed as a percentage change. Samples taken at 4 days after the last tamoxifen injection in mice were set to 100%. Data are shown for two mice from Experiment 6 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.t001" target="_blank">Table 1</a>; mouse S2 with low Arg1 enzyme activity and rescued mouse S4 with high Arg1 activity; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.g008" target="_blank">Fig 8D</a>) and three mice from Experiment 7 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.t001" target="_blank">Table 1</a>; non-rescued mouse S8 with high Arg1 activity, rescued mouse S9 with high Arg1 activity, and rescued mouse S11 with moderate Arg1 activity; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.g008" target="_blank">Fig 8D</a>) are shown.</p

A low-protein diet, with or without sodium phenylbutyrate (drug treated), minimally extends lifespan of mice with induced arginase-1 deficiency.

<p>(A) M = male (n = 12, divided into four groups of 3), F = female (n = 12, divided into four groups of 3). (B) Food consumption on a low-protein diet with sodium phenylbutyrate (drug treated) or without (control, non-drug treated) tends to wane prior to humane euthanization. Each bar within a group represents an individual day from Day +4 (left side) to Day +17 (right side). (C) Plasma levels of five metabolites at endpoint in three groups of non-drug treated mice on a low-protein diet. (D) Typical body weight loss is observed in same mice used in (B). Weight loss at humane endpoint was significantly lower (*, <i>p</i><0.05) than at Day +4.</p

Experiments to test AAV rescue of inducible arginase-1 deficient mice.

<p>Notes:</p><p>*Age of mice is based on the day of the first tamoxifen administration.</p><p>Timing of viral administration is relative to the last dose of tamoxifen (designated 0) and is given in days (-14 refers to two weeks prior to the last dose of tamoxifen, while +7 refers to one week after the last dose. Several extra vehicle-injected control mice were included in the studies (not shown in Table) to exclude viral-mediated toxicity.</p><p>^#Mouse A2 was euthanized at Day +21, even though healthy (within 2% of starting body weight and expressing 75% of control liver arginase activity; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.g008" target="_blank">Fig 8D</a>.</p><p>^!Mouse died due to non-viral mediated testicular morbidity.</p><p>CB7, hybrid cytomegalovirus enhancer/chicken β-actin promoter; TBG, thyroxine-binding globulin promoter. Mouse designation corresponds with data found in Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.g008" target="_blank">8C</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.g008" target="_blank">8D</a>.</p><p>^♂, male;</p><p>^♀, female.</p><p>Experiments to test AAV rescue of inducible arginase-1 deficient mice.</p