Fig 1.
Floxed Arg1-Cre mice (8–12 weeks old) treated with the standard tamoxifen dosing regimen (A) show evidence of reduced liver arginase-1 enzyme activity and protein (n = 3 all points, except Day +12 where n = 4) over time (B).
Day 0 is counted as the last day of tamoxifen administration, while Day +13 is the usual day for humane euthanization. Each Western blot lane (lower blot) corresponds to the enzyme activity bar below, with the exception that there is one extra lane in the blot corresponding to a mouse euthanized on Day +14. Image J quantitation relative to Day -2 is shown below the blot. The upper blot is the loading control for β-actin (β-Act). Enzyme activity was significantly decreased (*, p<0.05) compared to the “baseline” measurement (Day -2). (C) Typical phenotypic pattern of weight loss induced by Arg1 deficiency (n = 7 male; n = 5 female). Weight loss at humane endpoint was always significantly lower (p<0.05) than at Day +4. (D) Plasma ammonia in the same groups of mice used for protein and enzyme activity analysis. Day +12 levels were elevated but not significantly (p = 0.12).
Fig 2.
Ornithine supplementation does not rescue the induced arginase-1 deficiency phenotype.
(A) Citrulline (Cit), ornithine (Orn), arginine (Arg) and methionine (Met) plasma concentrations are similar in control and ornithine-treated groups of 4 week old mice at baseline (n = 4). (B) Ornithine-treated mice (●, n = 4) indeed show elevations in plasma ornithine, which demonstrates the efficacy of the protocol, and have significant increases in arginine compared to control mice (■,n = 5) *, p<0.05 relative to control. (C) Mice in both groups show similar patterns of weight loss (surrogate marker of health status). Weight loss at humane endpoint was always significantly lower (*, p<0.05) than at Day +4.
Fig 3.
A low-protein diet, with or without sodium phenylbutyrate (drug treated), minimally extends lifespan of mice with induced arginase-1 deficiency.
(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 (*, p<0.05) than at Day +4.
Fig 4.
Transgenic mouse rescue strategy for Arg1 inducible knockout mice.
(A) Generation of conditional Rosa 26 (R26)-promoter-based expression of Arg1, based upon ref [16]. Various primer sets used for genotyping and PCR products generated are shown. Exons (shaded rectangles), loxP sites (shaded triangles), selection cassette (hexagon) and transgene (shaded box) are depicted. SA, splice acceptor. (B,C) Genotypes of germline-transmitted cross-bred mice at R26 and Arg1 loci used to test inducible Arg1 transgene rescue of induced knockout of endogenous Arg1. Neonatal mice of 4 genotypes (n = 25, divided into groups as indicated) were administered tamoxifen on the day of birth and one day later, intragastrically, to generate the various Cre-excised alleles. (B) Representative PCR genotyping of each group (Grp). *, R26-Arg1 transgene positive;-, R26-Arg1 transgene negative with primers P2A/P2B, (R lane of each group). 195 bp band is Arg1Δ exon 7/8 Cre-excised allele, 252 bp band is residual Arg1fl allele after tamoxifen, and 1.2 kb band from Arg1 wild-type allele (using primers A,B,C described in ref. 10; left lane each group). (C) Mice carrying different allelic combinations at the R26 (left of /) and Arg1 (right of /) genetic loci were cross-bred to yield mice of various genotypes (only 4 shown and studied). One mouse from Group 2 died from gastric injury at Day +8 as observed on autopsy examination. (D) Liver Arg1 enzyme activity (n = 3–4) at Day +13 (Group 1) or Day +25 (Groups 2–4), along with some C57BL/6 control mice of similar ages; *, p<0.05.
Fig 5.
Real time quantitative PCR expression of Arg1.
Brain (A), kidney (B), and liver (C) of control C57BL/6 (n = 3), R26-Arg1 transgenic (TG; n = 3–5), and Arg1Δ knockout (KO; n = 3–5) mice.
Fig 6.
Measurement of plasma arginine (A), guanidinoacetic acid (B), and ornithine in tamoxifen-treated R26-Arg1 transgenic (TG) and non-transgenic mice both on the Arg1 KO background at three timepoints.
#, p<0.05 when compared to Day +4. Ornithine levels differed significantly between groups (n = 5–7; *, p<0.05).
Fig 7.
Generation of adeno-associated viral (AAV) vectors used in studies to rescue induced Arg1 deficiency.
(A) Maps (not to scale) of two AAV constructs used and the packaging serotypes (AAV8 and rh10). ITR, inverted terminal repeat; TBG, thyroxine-binding globulin; CB7-CI, hybrid cytomegalovirus enhancer/chicken β-actin promoter (CB7), along with a chicken β-actin intron (CI); WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; bGH-pA, bovine growth hormone polyA adenylation signal sequence; rBG, rabbit beta-globin polyA adenylation signal sequence. (B) Western blot indicating level of expression in livers of 4 separate mice (lanes 1–4) of AAV-expressed Arg1-eGFP relative to WT Arg1 and tubulin loading control post-tamoxifen (Day +13;4-week old mice injected with 2x1011 gc of the AAV8 construct shown in panel A) on Day -4. Lane 5 represents normal level of expression of Arg1 (and tubulin) in a mouse not injected with tamoxifen. In order to observe sufficient signal of the inducible Arg1 knockout band of lanes 1–4, the signal becomes over-saturated when detecting normal endogenous levels of Arg1 (lane 5). (C) Images of liver sections (i-iii) and dissociated hepatocytes (iv, v) from AAV8.TBG.PI.Arg1-eGFP.WPRE.bGH injected 4-week old mice 1 week after virus administration. Strong fluorescence signal from the fusion transgene is observed in the cytoplasm of some periportal hepatocytes (ii,iii) and also in dissociated cells (v) with corresponding phase contrast images (i, iv).
Table 1.
Experiments to test AAV rescue of inducible arginase-1 deficient mice.
Fig 8.
Some male mice are rescued from the lethal consequences of induced Arg1 deletion by AAVrh10.CB7.CI.Arg1-eGFP.WPRE.rBG administration.
(A) Survival of tamoxifen-treated male (n = 24) and female (n = 10) induced Arg1 KO mice injected with AAV vector (Expts 4–7, Table 1). 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) In situ 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 (Table 1). 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).
Fig 9.
Transgenic expression of Arg1-eGFP rescues some male Arg1 knockout mice but variably corrects plasma arginine.
(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 7.5x1010 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 (Table 1; mouse S2 with low Arg1 enzyme activity and rescued mouse S4 with high Arg1 activity; see Fig 8D) and three mice from Experiment 7 (Table 1; non-rescued mouse S8 with high Arg1 activity, rescued mouse S9 with high Arg1 activity, and rescued mouse S11 with moderate Arg1 activity; see Fig 8D) are shown.