scholarly journals Human Factor VIII Expression and Normalization of Bleeding Following AAV Gene Therapy in a Double Knockout Mouse Model of Hemophilia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3239-3239
Author(s):  
Stuart Bunting ◽  
Lening Zhang ◽  
Lin Xie ◽  
Sherry Bullens ◽  
Rajeev Mahimkar ◽  
...  
Keyword(s):  
Blood Loss ◽  
Factor Viii ◽  
Knockout Mouse ◽  
Bleeding Time ◽  
Wild Type ◽  
Double Knockout ◽  
Post Dose ◽  
Knockout Mouse Model ◽  
Protein Levels ◽  
Deficient Mice

Abstract Hemophilia A is an X-linked bleeding disorder caused by mutations in the gene encoding the Factor VIII coagulation protein (FVIII). Bleeding episodes in patients are reduced by prophylactic therapy or treated acutely using recombinant or plasma derived FVIII. Recently, Nathwani et al demonstrated in preclinical and clinical studies sustained expression of coagulation factor IX using AAV8 technology to deliver the human gene to the liver, driven by a liver specific promoter. The same group demonstrated FVIII expression in mice and primates using a modified B-domain truncated form of FVIII delivered in an AAV8 capsid. We have made an AAV5 construct containing a B-domain deleted FVIII gene (AAV5-SQ) with a liver specific promoter and evaluated it in a double knockout mouse model of hemophilia. The double knockout mice (DKO) were created by crossing factor VIII deficient mice with RAG2 deficient mice (RAG2 KO). The RAG2 KO mice lacked the ability to mount an adaptive immune response thereby allowing sustained expression of a human protein without the development of an antibody response. Eight week old male DKOs were randomly distributed into three groups, twenty per group, and treated via a single IV injection with either vehicle, or AAV5-SQ at 2 x 1013 or 1 x 1014 vg/kg. C57BL/6J mice comprised a fourth group and were treated with vehicle via a single IV injection to demonstrate wild type bleeding times and blood loss. Bleeding times and blood loss were assessed in these mice 8 weeks post-dose, at 16 weeks of age. In addition, forty 16 week old DKO mice were randomly divided into two groups and treated with a single IV injection of rhFVIII-SQ protein (rhSQ, Xyntha®) at either 50 or 200 IU/kg. Bleeding times were assessed in these mice 30 minutes post-dose, at 16 weeks of age. Eight weeks post dosing with either AAV5-SQ or vehicle, the tail bleeding time and blood loss were measured following transection of the tip of the tail for evaluation of the functional efficacy of AAV5-SQ gene therapy. Wild-type mice receiving vehicle had a mean of 0.040 ± 0.073 g blood loss and 5.11 ± 5.61 min bleeding time. DKO mice treated with vehicle had a mean blood loss and bleeding time of 0.741 ± 0.207 g and 28.96 ± 1.40 min, respectively. Mice receiving AAV5-SQ at 2x1013 vg/kg showed significantly reduced blood loss (0.387 ± 0.384 g, p=0.0008 vs DKO+ vehicle; p=0.0003 vs WT) and bleeding time (17.12 ± 11.58 min, p=0.00005 vs DKO+ vehicle; p=0.0013 vs WT) while 1x1014 vg/kg AAV5-SQ treatment corrected blood loss and bleeding times to wild-type levels (0.104 ± 0.203 g [p=0.192 vs WT, p= 5.49x10-12 vs DKO + vehicle] and 5.58 ± 9.32 mins [p=0.847 vs WT, p= 1.75x-13 vs DKO + vehicle], respectively). The effect of AAV5-SQ treatment on blood loss and bleeding time was comparable to the effects of rhSQ. DKO mice receiving 50 IU/kg of rhSQ had a mean blood loss and bleeding time of 0.492 ± 0.297 g and 18.14 ± 9.39 min, respectively, which was not significantly different from mice receiving AAV5-SQ at 2x1013 (p=0.343 for blood loss, p=0.760 for bleeding time). DKO mice receiving 200 IU/kg of rhSQ had a mean blood loss and bleeding time of 0.134 ± 0.191 g and 4.29 ± 6.16 min, respectively, which was not significantly different from mice receiving AAV5-SQ at 1x1014 (p=0.635 for blood loss, p=0.608 for bleeding time). In a separate experiment, 4 groups of DKO mice, n=10 per group, were injected with either vehicle, AAV5-SQ at 2x1013, AAV5-SQ at 2x1014 vg/kg or rhSQ at 50 IU/kg. Blood was collected 8 weeks after AAV5-SQ treatment or 5 and 30 min after rhSQ for evaluation of plasma hFVIII-SQ protein levels and activity. Expressed hFVIII-SQ levels were measured by electrochemiluminescence assay. Factor VIII-SQ protein levels at 2x1013 vg/kg AAV5-SQwere 46.8±44.0 ng/ml and 355±166ng/ml at 2x1014 vg/kg. At 50 IU/kg of rhSQ the plasma protein levels were 79.1±11.3 ng/ml at 5 min and 44.7±16.6 ng/ml at 30min post dosing. Western blot analysis of the plasma from these mice showed the expressed protein to be similar in size to rhSQ. In summary, AAV5-SQ injected into DKO hemophilic mice resulted in a dose dependent expression of B-domain deleted FVIII protein and a corresponding correction of bleeding time and blood loss. At the highest dose tested complete correction was achieved. Similar corrections in bleeding were observed at approximately the same plasma levels of FVIII protein produced either endogenously by AAV5-SQ or following exogenous administration of B-domain deleted FVIII. Disclosures Bunting: BioMarin Pharmaceutical: Employment. Zhang:BioMarin Pharmaceutical: Employment. Xie:BioMarin Pharmaceutical: Employment. Bullens:BioMarin Pharmaceutical: Employment. Mahimkar:BioMarin Pharmaceutical: Employment. Fong:BioMarin Pharmaceutical: Employment. Sandza:BioMarin Pharmaceutical: Employment. Colosi:BioMarin Pharmaceutical: Employment. Long:BioMarin Pharmaceutical: Employment. Vehar:BioMarin Pharmaceutical: Employment. Carter:BioMarin Pharmaceutical: Employment.

scholarly journals VWF-FVIII Interactions Influence Hemostatic Thrombus Stability in Murine Models of Hemophilia Α and Type 2N VWD

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1403-1403
Author(s):  
Laura L Swystun ◽  
Courtney Dwyer ◽  
Kate Nesbitt ◽  
Kassandra Hebert ◽  
David Lillicrap
Keyword(s):  
Blood Loss ◽  
Research Funding ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Wild Type ◽  
Bleeding Events ◽  
Experimental Conditions ◽  
Total Blood ◽  
Tail Vein ◽  
Deficient Mice

Abstract von Willebrand factor (VWF) and factor VIII (FVIII) circulate in the plasma as a non-covalent complex. VWF can influence FVIII activity by stabilizing FVIII plasma levels and transporting FVIII to the site of thrombus formation. We have previously generated a murine model of type 2N VWD through hydrodynamic expression of the severe R816W VWF variant that exhibits a 90% decrease in FVIII-binding ability and a failure to stabilize endogenous FVIII when expressed in a VWF-deficient mouse. Intravital studies of arteriole platelet thrombus formation demonstrated that FVIII knockout and type 2N VWD mice produced smaller, less stable thrombi. Aim: In this study, we assess the contribution of VWF and FVIII interactions to the formation and stability of hemostatic thrombi in a murine tail vein transection (TVT) bleeding model. Method: Under isoflurane anaesthesia the left lateral tail vein was transected using standardized measuring and transection templates (provided by Novo Nordisk, described in detail in Johansen et al. Hemophilia. 2016). Bleeding time was observed over the course of 60 minutes. Blood was collected into saline for hemoglobin quantification. For experimental conditions, 300 U/Kg wild type or type 2N (R816W) VWF was administered IV to VWF KO mice 2 hours prior to TVT in order to allow for endogenous FVIII stabilization. Results: Consistent with previous reports, VWF (44.15 min; p<0.0001) and FVIII-deficient mice (45 min; p<0.0001) exhibited a significantly prolonged total bleeding time relative to normal mice (4.6 min). Infusions of plasma-derived murine VWF (FVIII-free) into VWF deficient mice have demonstrated that wild type (WT) VWF is capable of restoring FVIII:C levels to ~75% 2 hours post-infusion, while the R816W variant does not influence FVIII stabilization and levels remain at ~15%. Mice infused with WT VWF had a shorter bleeding time (16.2 min) relative to mice infused with the severe type 2N VWD variant (44.67 minutes, p<0.0001). For FVIII KO mice, the primary bleeding time (1.53 min), defined as the period of time until first bleeding cessation, was not different from normal mice (1.2 min) but significantly extended for VWF KO mice (35.97 min, p<0.0001). Primary bleeding time was markedly reduced for VWF KO mice infused with WT VWF (4.97 min, p=0.07) confirming the restoration of primary hemostasis by infusion of murine plasma-derived VWF. Hemoglobin quantification confirmed that FVIII (243.8 mg, p<0.0001) and VWF KO mice (198.8 mg, p<0.0001) had increased blood loss compared to normal mice (9.91 mg). Similarly, VWF KO mice infused with type 2N VWF (308.04 mg) had increased blood loss compared to mice infused with WT VWF (73.78 mg, p=0.0008). Total blood loss and bleeding times were positively correlated across all groups (r2=0.625, p<0.0001). Thrombus stability was characterized by the frequency of spontaneous re-bleeding events. 11% of normal mice experienced one or more spontaneous re-bleeding events during the course of injury, while 100% of FVIII deficient mice experienced spontaneous re-bleeding (p=0.0004). A similar trend was observed for VWF KO mice infused with WT VWF (0%) and type 2N VWF (66.7%, p=0.021). Conclusions: These studies suggest that in the murine TVT model of physiological hemostasis, the initial hemostatic thrombus formation is predominantly driven by platelet plug formation and is VWF-dependent. In contrast, thrombus stability over the course of the model is reliant on the processes responsible for fibrin formation, and is FVIII-dependent. This suggests that patients with type 2N VWD and hemophilia A may have bleeding that is the result of impaired stabilization of the primary platelet plug. Disclosures Lillicrap: Baxalta: Research Funding; Biogen-Idec: Research Funding; Bayer: Research Funding; Octapharma: Research Funding.

scholarly journals Altered V-ATPase expression in renal intercalated cells isolated from B1 subunit-deficient mice by fluorescence-activated cell sorting

2013 ◽  
Vol 304 (5) ◽  
pp. F522-F532 ◽  
Author(s):  
Luca Vedovelli ◽  
John T. Rothermel ◽  
Karin E. Finberg ◽  
Carsten A. Wagner ◽  
Anie Azroyan ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Collecting Duct ◽  
Egfp Expression ◽  
Intercalated Cells ◽  
Wild Type ◽  
Western Blots ◽  
Atpase Subunit ◽  
Protein Levels ◽  
Baseline Conditions ◽  
Deficient Mice

Unlike human patients with mutations in the 56-kDa B1 subunit isoform of the vacuolar proton-pumping ATPase (V-ATPase), B1-deficient mice (Atp6v1b1−/−) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V-ATPases containing the alternative B2 subunit isoform into the apical membrane of renal medullary collecting duct intercalated cells (ICs). We previously reported that quantitative Western blots (WBs) from whole kidneys showed similar B2 protein levels in Atp6v1b1−/− and wild-type mice (Păunescu TG, Russo LM, Da Silva N, Kovacikova J, Mohebbi N, Van Hoek AN, McKee M, Wagner CA, Breton S, Brown D. Am J Physiol Renal Physiol 293: F1915–F1926, 2007). However, WBs from renal medulla (including outer and inner medulla) membrane and cytosol fractions reveal a decrease in the levels of the ubiquitous V-ATPase E1 subunit. To compare V-ATPase expression specifically in ICs from wild-type and Atp6v1b1−/− mice, we crossed mice in which EGFP expression is driven by the B1 subunit promoter (EGFP-B1+/+ mice) with Atp6v1b1−/− mice to generate novel EGFP-B1−/− mice. We isolated pure IC populations by fluorescence-assisted cell sorting from EGFP-B1+/+ and EGFP-B1−/− mice to compare their V-ATPase subunit protein levels. We report that V-ATPase A, E1, and H subunits are all significantly downregulated in EGFP-B1−/− mice, while the B2 protein level is considerably increased in these animals. We conclude that under baseline conditions B2 upregulation compensates for the lack of B1 and is sufficient to maintain basal acid-base homeostasis, even when other V-ATPase subunits are downregulated.

scholarly journals Molecular Alterations in the Stomach of Tff1-Deficient Mice: Early Steps in Antral Carcinogenesis

2020 ◽  
Vol 21 (2) ◽  
pp. 644 ◽  
Author(s):  
Eva B. Znalesniak ◽  
Franz Salm ◽  
Werner Hoffmann
Keyword(s):  
Cysteine Residue ◽  
Molecular Forms ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Rt Pcr ◽  
Molecular Alterations ◽  
Protein Levels ◽  
Increased Sensitivity ◽  
A Minor ◽  
Deficient Mice

TFF1 is a peptide of the gastric mucosa co-secreted with the mucin MUC5AC. It plays a key role in gastric mucosal protection and repair. Tff1-deficient (Tff1KO) mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas. Thus, these mice represent a model for gastric tumorigenesis. Here, we compared the expression of selected genes in Tff1KO mice and the corresponding wild-type animals (RT-PCR analyses). Furthermore, we systematically investigated the different molecular forms of Tff1 and its heterodimer partner gastrokine-2 (Gkn2) in the stomach (Western blot analyses). As a hallmark, a large portion of murine Tff1 occurs in a monomeric form. This is unexpected because of its odd number of seven cysteine residues. Probably the three conserved acid amino acid residues (EEE) flanking the 7th cysteine residue allow monomeric secretion. As a consequence, the free thiol of monomeric Tff1 could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. Furthermore, a minor subset of Tff1 forms a disulfide-linked heterodimer with IgG Fc binding protein (Fcgbp). Of special note, in Tff1KO animals a homodimeric form of Gkn2 was observed. In addition, Tff1KO animals showed strongly reduced Tff2 transcript and protein levels, which might explain their increased sensitivity to Helicobacter pylori infection.

scholarly journals Cooperation of Antiporter LAT2/CD98hc with Uniporter TAT1 for Renal Reabsorption of Neutral Amino Acids

2018 ◽  
Vol 29 (6) ◽  
pp. 1624-1635 ◽  
Author(s):  
Clara Vilches ◽  
Emilia Boiadjieva-Knöpfel ◽  
Susanna Bodoy ◽  
Simone Camargo ◽  
Miguel López de Heredia ◽  
...  
Keyword(s):  
Amino Acids ◽  
Knockout Mouse ◽  
Functional Relationship ◽  
Tubular Reabsorption ◽  
Compensatory Mechanisms ◽  
Double Knockout ◽  
Knockout Mouse Model ◽  
Relationship Of

Background Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter y+LAT1/CD98hc (SLC7A7/SLC3A2), which mediates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooperation between TAT1 and LAT2/CD98hc has been suggested by in vitro studies but has not been evaluated in vivo.Methods To study the functional relationship of TAT1 and LAT2/CD98hc in vivo, we generated a double-knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice).Results Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of y+LAT1/CD98hc.Conclusions The LAT2/CD98hc and TAT1 transporters functionally cooperate in vivo, and y+LAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA transporters may explain the lack of basolateral neutral aminoacidurias in humans.

scholarly journals 226: Genes or environment? A novel double knockout mouse model for fetal origins of autism study

2016 ◽  
Vol 214 (1) ◽  
pp. S135
Author(s):  
Hind N. Moussa ◽  
Baha M. Sibai ◽  
Sean C. Blackwell ◽  
David A. Fournie ◽  
Alejandra E. Ontiveros ◽  
...  
Keyword(s):  
Mouse Model ◽  
Knockout Mouse ◽  
Double Knockout ◽  
Fetal Origins ◽  
Knockout Mouse Model ◽  
Double Knockout Mouse

Monocyte-Derived Microparticles Improve Hemostasis in Factor VIIIDeficient Mice: Exploring the Role of PSGL-1/P-Selectin.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3386-3386
Author(s):  
Peter L. Gross ◽  
Nima Vaezzadeh ◽  
Lori Ivicic ◽  
Ran Ni ◽  
Bruno Esposito ◽  
...  
Keyword(s):  
Blood Loss ◽  
High Speed ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Wild Type ◽  
Spontaneous Bleeding ◽  
Platelet Accumulation ◽  
Deficient Mice ◽  
Tail Clip

Abstract Introduction: Microparticles derived from leukocytes contribute to fibrin formation at thrombi in vivo and factor VIII-deficient (FVIII) mice treated with an agent that elevates their microparticles have decreased bleeding. A novel therapy for hemophilia patients with inhibitors is needed. We evaluated whether microparticles generated in vitro could improve hemostasis in FVIII mice. Methods: Mouse CD11b positive monocytes, isolated by MACS, or cultured monocytic cells (WEHI274.1) were treated with the calcium ionophore A23187. The resulting microparticles (isolated by differential centrifugation, and defined as CD18 positive events less than 1 μm diameter) or PIPES buffer were infused intravenously into FVIII-deficient mice (B6.129S4-F8tm1Kaz) or control wild type B6.129 mice prior to evaluation. The amount of platelets in laser-generated thrombi in cremaster muscle arterioles was evaluated using high-speed intravital fluorescence microscopy. The amount of hemoglobin shed from a 2 mm tail tip amputation measured blood loss. Results: Infusion of MPs at doses above 1000/g resulted in the death of wild type mice; FVIII-deficient mice tolerated MPs at doses up to 4000/g. Blood loss after tail clip in FVIII-deficient mice was 6-fold higher than blood loss from wild type mice. Blood loss after tail clip in FVIII-deficient mice was reduced to normal after the infusion of MPs at concentrations as low as 400/g. MPs, at 400/g, from CD11b positive cells isolated from wild type, FVIII-deficient mice or PSGL-1-deficient mice all similarly reduced blood loss after tail clip in FVIII-deficient mice. The biological half life of MP effect on tail-bleeding was 3 hours. Platelet accumulation in cremaster arteriolar thrombi was impaired in FVIII-deficient mice. Infusion of MPs at a concentration of 1000/g normalized platelet accumulation, but infusion of MPs at a lower concentration (400/g) did not. Conclusion: Abnormal hemostasis in FVIII-deficient mice can be temporarily reversed by the infusion of in vitro generated monocyte-derived MPs, including MPs derived from monocytes from FVIII-deficient or PSGL-1-deficient mice. The dose whereby MPs normalize FVIII-deficient mice is different between the hemostasis and thrombosis models. To explore whether P-selectin at injuries is required for the effect of MPs, we have generated by cross-breeding FVIII/P-selectin double deficient mice. These mice are born at expected mendelian frequency. Two of 20 male FVIII/P-selectin double deficient mice had spontaneous bleeding at 8 weeks of age, one in the thigh and one from the ear. FVIII/P-selectin double deficient mice also have prolonged tail bleeding times, which will serve as a model for testing the P-selectin targeting of microparticles.

scholarly journals Correction of a murine model of von Willebrand disease by gene transfer

Blood ◽  
2006 ◽  
Vol 108 (3) ◽  
pp. 862-869 ◽  
Author(s):  
Robert G. Pergolizzi ◽  
Guangchun Jin ◽  
Diane Chan ◽  
Lorraine Pierre ◽  
James Bussel ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Bleeding Time ◽  
Active Form ◽  
Von Willebrand Disease ◽  
Wild Type ◽  
Bleeding Events ◽  
Specific Staining ◽  
Spontaneous Bleeding ◽  
Von Willebrand

Abstract von Willebrand disease (VWD), the most common inherited bleeding disorder in the U.S. population, is caused by defects in the expression and processing of von Willebrand factor (VWF), a blood glycoprotein required for normal hemostasis that mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet glycoproteins and to constituents of exposed connective tissue. To assess whether VWF deficiency can be corrected by gene transfer, a plasmid expressing the intact 8.4-kb murine VWF coding sequence, directed by the cyto-megalovirus immediate/early promoter/enhancer, was delivered through hydrodynamic tail vein injection into VWF knockout mice (VWF–/–) that exhibit defects in hemostasis, including highly prolonged bleeding time and spontaneous bleeding events, closely mimicking severe human VWD. VWF antigen levels in plasma from animals receiving VWF cDNA, but not control animals, revealed normalized levels of circulating VWF that persisted for at least 1 week after injection. Western blot analysis of plasma from animals receiving VWF cDNA, but not control animals, revealed high molecular–weight multimers with patterns similar to those observed in wild-type mice. Reverse transcription–polymerase chain reaction (RT-PCR) on RNA isolated from the livers of animals receiving VWF cDNA, but not control animals, demonstrated that VWF was expressed in the liver, and immunohistochemical analysis of the livers of treated VWF–/– mice revealed VWF-specific staining throughout the liver parenchyma but not in endothelial cells. Plasma from treated VWF–/– mice, but not control VWF–/– mice, supported the hypothesis that murine platelets aggregate in the presence of botrocetin. Although levels of circulating factor VIII in untreated VWF–/– mice were less than 10% those in wild-type mice, levels of factor VIII in VWF–/– animals treated with VWF cDNA, but not in control animals, were normalized to values in wild-type mice, indicating the restoration of factor VIII carrier function for VWF in treated mice that persisted for at least 1 week at higher doses of VWF cDNA. Most important, bleeding time was normalized by 48 hours after the delivery of VWF cDNA, but not by the control plasmid. These data suggest that with the use of gene transfer of VWF cDNA, VWF protein can be expressed, processed, and secreted in a physiologically active form; thus, it may be possible to correct VWD using gene transfer.

scholarly journals Disassociation of insulin action and Akt/FOXO signaling in skeletal muscle of older Akt-deficient mice

2012 ◽  
Vol 303 (11) ◽  
pp. R1186-R1194 ◽  
Author(s):  
Thomas H. Reynolds ◽  
Erin Merrell ◽  
Nicholas Cinquino ◽  
Megan Gaugler ◽  
Lily Ng
Keyword(s):  
High Fat Diet ◽  
Insulin Action ◽  
Mrna Levels ◽  
Wild Type ◽  
Akt Phosphorylation ◽  
Protein Levels ◽  
Akt Isoforms ◽  
Forkhead Box ◽  
Gene Ablation ◽  
Deficient Mice

The purpose of the present study was to determine the effect of Akt gene ablation on Akt/Forkhead Box O (FOXO) signaling and atrogene expression. This was accomplished by studying wild-type (WT) and isoform-specific Akt knockout (Akt1−/− and Akt2−/−) mice. The ability of insulin to promote Akt phosphorylation on Ser473 was significantly lower in extensor digitorum longus (EDL) and soleus muscles from Akt1−/− and Akt2−/− mice compared with WT mice. Total Akt1 protein levels were significantly lower in EDL muscles of Akt2−/− mice compared with WT mice, a process that appears to be posttranscriptionally regulated as Akt1 mRNA levels were unchanged. The loss of Akt1 protein in EDL muscles of Akt2−/− mice does not appear to be due to insulin resistance because 4 mo of a high-fat diet failed to reduce Akt1 protein levels in muscles of WT mice. Although FOXO3a phosphorylation and atrogin-1 expression were unaltered in muscles of Akt1−/− and Akt2−/− mice, the expression of the atrogenes Bnip3 and gabarapl were significantly elevated in muscles of both Akt1 and Akt2 knockout mice. Finally, the expression of striated activator of Rho signaling was significantly increased in muscles of Akt2−/− mice compared with Akt1−/− and WT mice. Our results demonstrate that the ablation of Akt isoforms disassociates insulin action and Akt/FOXO signaling to atrogenes.

scholarly journals Micro-MRI phenotyping of a novel double-knockout mouse model of congenital heart disease

2010 ◽  
Vol 12 (Suppl 1) ◽  
pp. P1 ◽  
Author(s):  
Jon O Cleary ◽  
Karen McCue ◽  
Anthony N Price ◽  
Sarah Beddow ◽  
Roger J Ordidge ◽  
...  
Keyword(s):  
Congenital Heart Disease ◽  
Heart Disease ◽  
Mouse Model ◽  
Knockout Mouse ◽  
Congenital Heart ◽  
Double Knockout ◽  
Knockout Mouse Model ◽  
Double Knockout Mouse

scholarly journals MFN1 and MFN2 Are Dispensable for Sperm Development and Functions in Mice

2021 ◽  
Vol 22 (24) ◽  
pp. 13507
Author(s):  
Junru Miao ◽  
Wei Chen ◽  
Pengxiang Wang ◽  
Xin Zhang ◽  
Lei Wang ◽  
...  
Keyword(s):  
Germ Cells ◽  
Knockout Mice ◽  
Seminiferous Tubules ◽  
Wild Type ◽  
Double Knockout ◽  
Wild Type Littermate ◽  
Mitofusin 2 ◽  
Sperm Development ◽  
Mitofusin 1 ◽  
Deficient Mice

MFN1 (Mitofusin 1) and MFN2 (Mitofusin 2) are GTPases essential for mitochondrial fusion. Published studies revealed crucial roles of both Mitofusins during embryonic development. Despite the unique mitochondrial organization in sperm flagella, the biological requirement in sperm development and functions remain undefined. Here, using sperm-specific Cre drivers, we show that either Mfn1 or Mfn2 knockout in haploid germ cells does not affect male fertility. The Mfn1 and Mfn2 double knockout mice were further analyzed. We found no differences in testis morphology and weight between Mfn-deficient mice and their wild-type littermate controls. Spermatogenesis was normal in Mfn double knockout mice, in which properly developed TRA98+ germ cells, SYCP3+ spermatocytes, and TNP1+ spermatids/spermatozoa were detected in seminiferous tubules, indicating that sperm formation was not disrupted upon MFN deficiency. Collectively, our findings reveal that both MFN1 and MFN2 are dispensable for sperm development and functions in mice.

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