control vector pcmv6  (OriGene)

Journal: Nature Communications

Article Title: Base-editing corrects metabolic abnormalities in a humanized mouse model for glycogen storage disease type-Ia

doi: 10.1038/s41467-024-54108-1

Figure Lengend Snippet: Newborn (NB) huR83C mice, non-fasted, were treated with a high dose of BEAM-301 (301H) at 1.5 mg/kg. At age 3 weeks, the phenotype of the resulting NB-301H mice (NB-301H-3W) was evaluated and compared to age-matched unedited huR83C (huR83C-3W), wild-type (mR83-3W), and heterozygote (mR83/huR83C-3W) mice. a Liver and kidney microsomal G6Pase-α activity. Liver (mR83-3W, n = 5; mR83/huR83C-3W, n = 11; huR83C-3W, n = 8; NB-301H-3W, n = 6) and kidney (mR83-3W, n = 6; mR83/huR83C-3W, n = 10; huR83C-3W, n = 8; NB-301H-3W, n = 6). b Restoration of hepatic G6Pase-α activity as a function of base editing efficiency in NB-301H-3W mice ( n = 6) along with on-target and bystander values of liver base editing. c Fasting blood glucose levels in control (mR83-3W and mR83/huR83C-3W; n = 5) and NB-301H-3W ( n = 5) mice. d Histochemical analysis of liver and kidney G6Pase-α activity in control (mR83-3W and mR83/huR83C-3W, n = 6), untreated (huR83C-3W, n = 6), and NB-301H-3W ( n = 6) mice. Each image represents an individual mouse. The arrow indicates the kidney cortex. Scale bar = 100 µm. The numbers represent hepatic G6Pase-α activity expressed in the mice. e Body weight (BW), liver weight (LW)/BW, and kidney weight (KW)/BW values of control (mR83-3W and mR83/huR83C-3W, n = 25), untreated (huR83C-3W, n = 17), and NB-301H-3W ( n = 6) mice. f Size comparison of mR83 and huR83C mice, showing growth retardation of the huR83C mice at age 3 weeks. g Restoration of hepatic G6Pase-α activity as a function of LW/BW values and hepatic levels of glycogen, triglyceride, and G6P in the NB-301H-3W mice ( n = 6). Statistics were performed using a two-tailed unpaired T test. Data are presented as Mean values ± SEM, and individual data points for each animal are displayed. * denotes p < 0.05, ** denotes p value < 0.005.

Article Snippet: A human cDNA encoding the open reading frame for G6PC1 - c.247C > T ( G6PC1 -R83C) were inserted into exon 1 of the mouse G6pc gene at the ATG start codon (Taconic Biosciences) in a way that created a premature STOP codon in the coding sequence of the mouse G6pc exon 1.

Techniques: Activity Assay, Control, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Base-editing corrects metabolic abnormalities in a humanized mouse model for glycogen storage disease type-Ia

doi: 10.1038/s41467-024-54108-1

Figure Lengend Snippet: Newborn (NB) huR83C mice, non-fasted, were treated with a high dose of BEAM-301 (301H) at 1.5 mg/kg. At age 3 weeks, biochemical phenotype of the edited mice was analyzed and compared to the age matched unedited huR83C. The 3-week-old mR83 and mR83/huR83C littermates displaying a wild-type phenotype were used as the controls. a Blood glucose levels (control-3W, n = 24; huR83C-3W, n = 16; NB-301H-3W, n = 6), serum cholesterol levels (control-3W, n = 16; huR83C-3W, n = 7; NB-301H-3W, n = 6), serum triglyceride levels (control-3W, n = 24; huR83C-3W, n = 16; NB-301H-3W, n = 6), serum lactate and uric acid levels (control-3W, n = 16; huR83C-3W, n = 8; NB-301H-3W, n = 6). b Liver glucose, triglyceride, lactate, glycogen, and G6P levels in control ( n = 16), huR83C-3W ( n = 8), and NB-301H-3W ( n = 6) mice. c Hematoxylin and eosin (H&E)-staining of liver and kidney sections in mR83/huR83C-3W ( n = 6), huR83C-3W ( n = 6), and NB-301H-3W ( n = 6) mice. The liver and kidney in all experimental animals were examined. A single image from an individual mouse, representative of the results in all mice, is shown to illustrate the results. Scale bar = 20 µm. The numbers represent hepatic G6Pase-α activity expressed in the mice. d Oil Red O staining of liver and kidney sections in mR83/huR83C-3W ( n = 6), huR83C-3W ( n = 6), and NB-301H-3W ( n = 6) mice. The liver and kidney in all experimental animals were examined. A single image from an individual mouse, representative of the results in all mice, is shown to illustrate the results. Scale bar = 20 μm. The numbers represent hepatic G6Pase-α activity expressed in the mice. Statistics were performed using a two-tailed unpaired T test. Data are presented as Mean values ± SEM, and individual data points for each animal are displayed. * denotes p < 0.05, ** denotes p value < 0.005.

Article Snippet: A human cDNA encoding the open reading frame for G6PC1 - c.247C > T ( G6PC1 -R83C) were inserted into exon 1 of the mouse G6pc gene at the ATG start codon (Taconic Biosciences) in a way that created a premature STOP codon in the coding sequence of the mouse G6pc exon 1.

Techniques: Control, Staining, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Base-editing corrects metabolic abnormalities in a humanized mouse model for glycogen storage disease type-Ia

doi: 10.1038/s41467-024-54108-1

Figure Lengend Snippet: Newborn (NB) and 3-week-old (3W) huR83C mice, non-fasted, were treated with 301H (BEAM-301 at 1.5 mg/kg) and the phenotype of the resulting NB-301H-8W and 3W-301H-8W mice was analyzed at age 8 weeks. The sex-matched mR83-8W and mR83/huR83C-8W littermates displaying a wild-type phenotype were used as the controls. a Liver and kidney microsomal G6Pase-α activity in mR83-8W ( n = 8), mR83/huR83C-8W ( n = 9), NB-301H-8W ( n = 8), and 3W-301H-8W ( n = 9) mice. b Restoration of hepatic G6Pase-α activity as a function of base editing efficiency along with on-target and bystander values of liver base editing. NB-301H-8W ( n = 8); 3W-301H-8W ( n = 9). c Fasting blood glucose levels in control ( n = 23), NB-301H-8W ( n = 8), and 3W-301H-8W ( n = 9) mice. d BW, LW/BW, and KW/BW values of control ( n = 23), NB-301H-8W ( n = 8), and 3W-301H-8W ( n = 9) mice. e Restoration of hepatic G6Pase-α activity as a function of LW/BW values and hepatic levels of glycogen and G6P in the 301H-8W mice ( n = 17), including NB-301H-8W ( n = 8) and 3W-301H-8W ( N = 9) mice. f Blood glucose and serum cholesterol, triglyceride, lactate, and uric acid levels in control ( n = 17), NB-301H-8W ( n = 8), and 3W-301H-8W ( n = 9) mice. g Liver glucose, glycogen, triglyceride, lactate, and G6P levels in control ( n = 17), NB-301H-8W ( n = 8), and 3W-301H-8W ( n = 9) mice. Statistics were performed using a two-tailed unpaired T test. Data are presented as Mean values ± SEM, and individual data points for each animal are displayed. * denotes p < 0.05, ** denotes p value < 0.005.

Article Snippet: A human cDNA encoding the open reading frame for G6PC1 - c.247C > T ( G6PC1 -R83C) were inserted into exon 1 of the mouse G6pc gene at the ATG start codon (Taconic Biosciences) in a way that created a premature STOP codon in the coding sequence of the mouse G6pc exon 1.

Techniques: Activity Assay, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Base-editing corrects metabolic abnormalities in a humanized mouse model for glycogen storage disease type-Ia

doi: 10.1038/s41467-024-54108-1

Figure Lengend Snippet: Newborn (NB) and 3-week-old (3W) huR83C mice, non-fasted, were treated with a low dose of BEAM-301 (301L) at 0.75 mg/kg and the phenotype of the resulting NB-301L-8W and 3W-301L-8W mice was analyzed at age 8 weeks. The sex-matched mR83 and mR83/hR83C littermates displaying a wild-type phenotype were used as the controls. a Liver microsomal G6Pase-α activity in control ( n = 17), NB-301L-8W ( n = 9), and 3W-301L-8W ( n = 8) mice. b Restoration of hepatic G6Pase-α activity as a function of base editing efficiency along with on-target and bystander values of liver base editing in NB-301L-8W ( n = 9) and 3W-301L-8W ( n = 8) mice. c Fasting blood glucose levels in control ( n = 17), NB-301L-8W ( n = 9), and 3W-301L-8W ( n = 8) mice. d BW, LW/BW, and KW/BW values of control ( n = 23), NB-301L-8W ( n = 9), and 3W-301L-8W ( n = 8) mice. e Restoration of hepatic G6Pase-α activity as a function of LW/BW values and hepatic levels of glycogen and G6P in the edited 301L-8W ( n = 17) mice, including NB-301L-8W ( n = 9) and 3W-301L-8W ( n = 8) mice. f Blood glucose and serum cholesterol, triglyceride, lactate, and uric acid levels in control ( n = 17), NB-301L-8W ( n = 9), and 3W-301L-8W ( n = 8) mice. g Liver glucose, glycogen, triglyceride, lactate, and G6P levels in control ( n = 17), NB-301L-8W ( n = 9), and 3W-301L-8W ( n = 8) mice. Statistics were performed using a two-tailed unpaired T test. Data are presented as Mean values ± SEM, and individual data points for each animal are displayed. * denotes p < 0.05, ** denotes p value < 0.005.

Article Snippet: A human cDNA encoding the open reading frame for G6PC1 - c.247C > T ( G6PC1 -R83C) were inserted into exon 1 of the mouse G6pc gene at the ATG start codon (Taconic Biosciences) in a way that created a premature STOP codon in the coding sequence of the mouse G6pc exon 1.

Techniques: Activity Assay, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Base-editing corrects metabolic abnormalities in a humanized mouse model for glycogen storage disease type-Ia

doi: 10.1038/s41467-024-54108-1

Figure Lengend Snippet: Newborn (NB) huR83C mice, non-fasted, were treated with 301H (BEAM-301 at 1.5 mg/kg) and the phenotype of the NB-301H-dosed mice (NB-301H-53W, n = 19) was evaluated at 53 weeks of age using sex-matched wild-type littermates (mR83-53W, n = 16) as the controls. a Liver microsomal G6Pase-α activity. b Restoration of hepatic G6Pase-α activity as a function of base editing efficiency along with on-target and bystander values of liver base editing. c Fasting blood glucose levels. d BW, LW/BW, and KW/BW values. e Restoration of hepatic G6Pase-α activity as a function of LW/BW values and hepatic levels of glycogen and G6P in the edited mice. f Blood glucose and serum cholesterol, triglyceride, lactate, and uric acid levels. g Liver glucose, glycogen, triglyceride, lactate, and G6P levels. Statistics were performed using a two-tailed unpaired T test. Data are presented as Mean values ± SEM, and individual data points for each animal are displayed. * denotes p < 0.05, ** denotes p value < 0.005.

Article Snippet: A human cDNA encoding the open reading frame for G6PC1 - c.247C > T ( G6PC1 -R83C) were inserted into exon 1 of the mouse G6pc gene at the ATG start codon (Taconic Biosciences) in a way that created a premature STOP codon in the coding sequence of the mouse G6pc exon 1.

Techniques: Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Base-editing corrects metabolic abnormalities in a humanized mouse model for glycogen storage disease type-Ia

doi: 10.1038/s41467-024-54108-1

Figure Lengend Snippet: Three-week-old (3W) huR83C mice, non-fasted, were treated with 301L (BEAM-301 at 0.75 mg/kg) and the phenotype of the 3W-301L-dosed mice (3W-301L-53W) was evaluated at 53 weeks of age using the sex-matched mR83-53W and mR83/huR83C-53W littermates (Control-53W) as the controls. a Liver microsomal G6Pase-α activity in Control ( n = 14) and 3W-301L-53W ( n = 9) mice. b Restoration of hepatic G6Pase-α activity as a function of base editing efficiency along with on-target and bystander values of liver base editing ( n = 9). c Fasting blood glucose levels in Control-53W ( n = 13) and 3W-301L-53W ( n = 8) mice. d BW, LW/BW, and KW/BW values in Control ( n = 14) and 3W-301L-53W ( n = 9) mice. e Restoration of hepatic G6Pase-α activity as a function of LW/BW values and hepatic levels of glycogen and G6P in the edited mice ( n = 9). f Blood glucose and serum cholesterol, triglyceride, lactate, and uric acid levels in Control ( n = 14) and 3W-301L-53W ( n = 9) mice. g Liver glucose, glycogen, triglyceride, lactate, and G6P levels (nmol/mg) in Control ( n = 14) and 3W-301L-53W ( n = 9) mice. Statistics were performed using a two-tailed unpaired T test. Data are presented as Mean values ± SEM, and individual data points for each animal are displayed. * denotes p < 0.05, ** denotes p value < 0.005.

Article Snippet: A human cDNA encoding the open reading frame for G6PC1 - c.247C > T ( G6PC1 -R83C) were inserted into exon 1 of the mouse G6pc gene at the ATG start codon (Taconic Biosciences) in a way that created a premature STOP codon in the coding sequence of the mouse G6pc exon 1.

Techniques: Control, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Base-editing corrects metabolic abnormalities in a humanized mouse model for glycogen storage disease type-Ia

doi: 10.1038/s41467-024-54108-1

Figure Lengend Snippet: Three-week-old (3W) huR83C mice, non-fasted, were treated with 301L (BEAM-301 at 0.75 mg/kg) and the biochemical phenotype of the 3W-301L-dosed mice (3W-301L-53W), was evaluated at 53 weeks of age using the sex-matched mR83-53W and mR83/huR83C-53W littermates (Control-53W) as the controls. a BMI values in Control-53W ( n = 14) and 3W-301L-53W ( n = 9) mice. b Insulin tolerance test in Control-53W ( n = 11) and 3W-301L-53W ( n = 8) mice. A reduced insulin dose of 0.25 IU/kg was used because GSD-Ia mice have an increased insulin sensitivity . c ALT and AST values in Control-53W ( n = 12), 3W-301L-53W ( n = 9), mR83-53W ( n = 15) and NB-301H-53W ( n = 17) mice. d Histochemical analysis of liver G6Pase-α activity in Control-53W ( n = 6) and 3W-301L-53W ( n = 6) mice. Each image represents an individual mouse. Scale bar = 100 µm. The numbers represent hepatic G6Pase-α activity expressed in the mice. e Hematoxylin and eosin (H&E) and Oil Red O staining of the liver sections in Control-53W ( n = 6) and 3W-301L-53W ( n = 6). The liver in the six pairs of experimental animals were examined. A single image from an individual mouse, representative of the results in all mice, is shown to illustrate the results. Scale bar = 20 µm. The numbers represent hepatic G6Pase-α activity expressed in the mice. Statistics were performed using a two-tailed unpaired T test. Data are presented as Mean values ± SEM, and individual data points for each animal are displayed. * denotes p < 0.05, ** denotes p value < 0.005.

Article Snippet: A human cDNA encoding the open reading frame for G6PC1 - c.247C > T ( G6PC1 -R83C) were inserted into exon 1 of the mouse G6pc gene at the ATG start codon (Taconic Biosciences) in a way that created a premature STOP codon in the coding sequence of the mouse G6pc exon 1.

Techniques: Control, Activity Assay, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Tissue-Specific Alteration of Metabolic Pathways Influences Glycemic Regulation

doi: 10.1101/790618

Figure Lengend Snippet: Related to Figure S4. (A) Protein expression levels of missense G6PC variants were determined in Huh7 cells (n=4-5) and (B) HEK293 cells (n=5) by western blot densitometric analysis of FLAG-tagged G6PC constructs relative to tubulin control, with representative blots shown. (C) Protein expression levels of PTV Q347X were determined in Huh7 cells (n=3) and (D) HEK293 cells (n=4) by western blot densitometric analysis of V5-tagged G6PC constructs relative to tubulin control, with representative blots shown. Bars in red indicate variants that are statistical drivers of the gene-based signal. (E) Cellular localisation of V5-tagged G6PC-Q347X was assessed in Huh7 cells and overlaid with markers for the ER (calreticulin) and the trans-golgi network (TGN46). White arrows point to positions of the Golgi apparatus. Scale bar indicates 10μm. (F) Glucose-6-phosphatase activity of unglycosylated WT G6PC protein obtained from tunicamycin-treated (Tuni) HEK293 microsomes (n=2), with representative western blot of microsomal protein shown. All data presented as mean ± SEM. * p=0.01-0.05; ** p=0.001-0.01; *** p<0.001.

Article Snippet: Human G6PC (NM_000151.3) and G6PC2 cDNA (NM_021176.2) within a pCMV6-Entry vector (with a C-terminal Myc-FLAG-tag) was purchased from OriGene (RC215623 and RC211146 respectively).

Techniques: Expressing, Western Blot, Construct, Activity Assay

Journal: bioRxiv

Article Title: Tissue-Specific Alteration of Metabolic Pathways Influences Glycemic Regulation

doi: 10.1101/790618

Figure Lengend Snippet: Related to Figure S5. (A) Expression levels of the glycosylated forms (upper bands only) of G6PC2 variant proteins were determined in INS-1 832/13 cells by western blot densitometric analysis of Myc-tagged G6PC2 constructs relative to tubulin control (n=5). Representative blots are shown for untreated cells together with cells treated with proteasomal inhibitor MG-132 or lysosomal inhibitor chloroquine. (B) Glucose-6-phosphatase activity of L173T and L173V variants in G6PC (proxy for I171T and I171V in G6PC2 respectively) in HEK293 against increasing glucose-6-phosphate concentrations (n=4), with mean Vmax ± SEM and Km ± SEM values shown for WT and each variant. (C) Glucose-6-phosphatase activity of F258L variant in G6PC (proxy for F256L in G6PC2) in HEK293 against increasing glucose-6-phosphate concentrations (n=3), with mean Vmax ± SEM and Km ± SEM values shown. Vmax and Km results were computed based on the Michaelis-Menten kinetic model. (D) Effect of G6PC2 WT and variant protein expression on luciferase activity driven by ER stress response elements in HEK293 cells. Relative luciferase units corrected for background activity were normalised to WT for each reporter, from n=6 across two independent experiments (except for F256L, n=3 in one experiment) using two-way ANOVA with Fisher’s LSD test comparing each variant to WT. (E) Cellular localisation of R283X in EndoC-βH1 overlaid with markers for the ER (calreticulin) and the trans-golgi network (TGN46). White arrows point to positions of the Golgi apparatus. Scale bar indicates 10μm. (F) Insulin secretion normalised to total content at basal and high glucose conditions (with and without drug treatments) following 96-120h G6PC2 knockdown in EndoC-βH1. Unpaired two-tailed Students’ t tests were used to compare G6PC2 knockdown to control for each condition, from n=16 across 4 independent experiments. Tol: tolbutamide; Diaz: diazoxide. All data presented as mean ± SEM. * p=0.01-0.05; ** p=0.001-0.01; *** p<0.001.

Article Snippet: Human G6PC (NM_000151.3) and G6PC2 cDNA (NM_021176.2) within a pCMV6-Entry vector (with a C-terminal Myc-FLAG-tag) was purchased from OriGene (RC215623 and RC211146 respectively).

Techniques: Expressing, Variant Assay, Western Blot, Construct, Activity Assay, Luciferase, Two Tailed Test