conditional col4a1 flex41 mutant mouse  (inGenious Targeting Laboratory)

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Skeletal pathology in mouse models of Gould syndrome is partially alleviated by genetically reducing TGFβ signaling

doi: 10.1016/j.matbio.2024.07.005

Figure Lengend Snippet: Altered skeletal development in Col4a1 + /Δex41 mice. (A) Col4a1 + /Δex41 mice exhibit reduced body size compared to their Col4a1 +/+ littermates throughout life (shown are postnatal day (P) 7 mice). (B-H) Representative images of skeletal preparations from P7 mice stained with Alcian blue and Alizarin red showing reduced ossification in (C-D) calvaria (black arrow indicate reduced Alizarin red staining), (E-F) ribs (black asterisks demarcate regions where alizarin red staining is absent in E and F), and (E-H) vertebrae (white asterisks mark the vertebral column in E and F) from Col4a1 + /Δex41 mice compared to sex-matched Col4a1 +/+ littermates. n = 3 mice per genotype. Shown are skeletal preparations from male mice, similar results were observed in female mice.

Article Snippet: The conditional Col4a1 Flex41 mutant mouse was generated by InGenious Targeting Laboratory (Stony Brook, NY) and previously characterized and validated [ , ].

Techniques: Staining

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Skeletal pathology in mouse models of Gould syndrome is partially alleviated by genetically reducing TGFβ signaling

doi: 10.1016/j.matbio.2024.07.005

Figure Lengend Snippet: Altered bone marker gene expression in P7 Col4a1 + /Δex41 mice. (A-B) qPCR analyses showing altered expression of bone marker genes in calvaria (A) and femurs (B) from P7 Col4a1 + /Δex41 mice compared to their Col4a1 +/+ littermates. Expression of bone marker genes was normalized to the expression of β2-microglobulin . Data are presented as fold expression relative to Col4a1 +/+ levels, mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001, unpaired two-tailed Student t -test. Since no difference in bone marker gene expression were observed between male and female mice, both sexes were combined with similar numbers of males and females included.

Article Snippet: The conditional Col4a1 Flex41 mutant mouse was generated by InGenious Targeting Laboratory (Stony Brook, NY) and previously characterized and validated [ , ].

Techniques: Marker, Gene Expression, Expressing, Two Tailed Test

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Skeletal pathology in mouse models of Gould syndrome is partially alleviated by genetically reducing TGFβ signaling

doi: 10.1016/j.matbio.2024.07.005

Figure Lengend Snippet: Morphometric analyses of femoral cortical and trabecular bone in Col4a1 + /Δex41 mice. (A-B) Representative 3D images generated from microcomputed tomography (μCT) analysis of cortical and trabecular parameters of femurs from 3 to 4 months old female (A) and male (B) Col4a1 +/+ and Col4a1 + /Δex41 mice. Scale bar=200μm. (C-J) Bone parameters of femurs analyzed by μCT. (C-E) Cortical bone parameters of the middle diaphysis. While cortical bone area fraction (Ct.Ar/T.Ar) (C) and cortical bone mineral density (BMD) (E) were not significantly different between genotypes, cortical bone thickness was significantly reduced in Col4a1 + /Δex41 mice compared to sex-matched Col4a1 +/+ littermates (D). (F-J) Femoral trabecular bone parameters. Trabecular bone volume fraction (BV/TV) (F) and trabecular number (G) were significantly reduced and spacing (H) was significantly increased both in female and male Col4a1 + /Δex41 mice compared to sex-matched Col4a1 +/+ littermates, while trabecular thickness was significantly reduced in male, but not female Col4a1 + /Δex41 mice (I). Trabecular BMD was also reduced in Col4a1 + /Δex41 mice compared to their sex-matched Col4a1 +/+ littermates (J). n = 8 for Col4a1 +/+ and Col4a1 + /Δex41 female mice and n = 9 and 7 for Col4a1 +/+ and Col4a1 + /Δex41 male mice, respectively. Data are presented as mean ± SD. * p ≤ 0.05, ** p < 0.01, *** p < 0.001, unpaired two-tailed Student t -test. Additional parameters are shown in .

Article Snippet: The conditional Col4a1 Flex41 mutant mouse was generated by InGenious Targeting Laboratory (Stony Brook, NY) and previously characterized and validated [ , ].

Techniques: Generated, Tomography, Two Tailed Test

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Skeletal pathology in mouse models of Gould syndrome is partially alleviated by genetically reducing TGFβ signaling

doi: 10.1016/j.matbio.2024.07.005

Figure Lengend Snippet: Altered bone mechanical properties in Col4a1 + /Δex41 mice. (A-D) Assessment of femoral bone mechanical properties by 3-point bending flexural test revealed a trend toward reduced femoral stiffness in Col4a1 + /Δex41 mice compared to sex-matched Col4a1 +/+ littermates (A), a significant reduction in yield force in female but not male Col4a1 + /Δex41 mice (B), a decrease in ultimate force that reached statistical significance in male but not female Col4a1 + /Δex41 mice (C), and a significant decrease in work in male but not female Col4a1 + /Δex41 mice (D). n = 4/group. Data are presented as mean ± SD. * p < 0.05; ** p < 0.01, unpaired two-tailed Student t -test. Additional details are shown in .

Article Snippet: The conditional Col4a1 Flex41 mutant mouse was generated by InGenious Targeting Laboratory (Stony Brook, NY) and previously characterized and validated [ , ].

Techniques: Two Tailed Test

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Skeletal pathology in mouse models of Gould syndrome is partially alleviated by genetically reducing TGFβ signaling

doi: 10.1016/j.matbio.2024.07.005

Figure Lengend Snippet: Morphometric analyses of femoral cortical and trabecular bones in Col4a1 + /G394V and Col4a1 + /G1344D mice. (A-B) Representative 3D images generated from μCT analysis of cortical and trabecular bone parameter of femurs from 3 to 4mo (A) female and (B) male Col4a1 +/+ , Col4a1 + /G394V , and Col4a1 + /G1344D mice. Scale bar=200μm. (C-J) Bone parameters of femurs analyzed by μCT. (C-E) Cortical bone parameters of the middle diaphysis, including cortical bone area fraction (Ct.Ar/T.Ar) (C), cortical bone thickness (D) and cortical BMD (E), were similar between Col4a1 + /mut mice and their Col4a1 +/+ littermates. (F-J) Femoral trabecular bone parameters. Trabecular bone density (BV/TV) (F) and trabecular numbers (G) are reduced, and trabecular separation (H) increased in Col4a1 + /mut mice compared to Col4a1 +/+ littermates. Femoral trabecular thickness (I) was slightly increased in Col4a1 + /G394V females compared to sex-matched Col4a1 +/+ and Col4a1 + /G1344D mice and a trend towards reduced BMD was observed in Col4a1 + /G394V mice compared to Col4a1 +/+ mice (J). n = 11, 5, and 10 for Col4a1 +/+ , Col4a1 + /G394V , and Col4a1 + /G1344D female mice, and n = 11, 6, and 8 for Col4a1 +/+ , Col4a1 + /G394V , and Col4a1 + /G1344D male mice, respectively. Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, one-way ANOVA. Additional parameters are shown in .

Article Snippet: The conditional Col4a1 Flex41 mutant mouse was generated by InGenious Targeting Laboratory (Stony Brook, NY) and previously characterized and validated [ , ].

Techniques: Generated

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Skeletal pathology in mouse models of Gould syndrome is partially alleviated by genetically reducing TGFβ signaling

doi: 10.1016/j.matbio.2024.07.005

Figure Lengend Snippet: Genetically reducing Tgfb1 levels partially improves structural bone parameters in Col4a1 + /Δex41 mice. Morphometric analyses of femurs from 3 to 4 mo male mice. (A-B) Representative 3D images generated from μCT analysis of cortical and trabecular femoral bones from 3 to 4mo untreated (NT), 4PBA-treated, and Tgfb1 heterozygous Col4a1 +/+ (A) and Col4a1 + /Δex41 (B) mice. Scale bar=200μm. (C-E) Cortical bone parameters of the middle diaphysis and (F-J) trabecular bone parameters analyzed by μCT showing significant reductions in cortical thickness, trabecular bone density and number and significant increase in trabecular separation in untreated Col4a1 + /Δex41 mice compared their Col4a1 +/+ counterparts (D, F, G, and H, respectively). 4PBA treatment increased trabecular bone volume fraction and cortical and trabecular thicknesses in Col4a1 +/+ mice (F, D, and I). Although 4PBA improved trabecular number and separation (G, H) in Col4a1 + /Δex41 mice, it significantly decreased trabecular bone thickness and BMD (I and J). While Tgfb1 heterozygosity significantly reduced cortical and trabecular thickness and tended to reduce cortical BMD and trabecular bone volume fraction in Col4a1 +/+ mice (D, I, E, and F), it significantly improved trabecular bone number and separation and tended to increase trabecular bone volume fraction in Col4a1 + /Δex41 mice (G, H, and F, respectively). n = 9, 10, 8 for untreated, 4PBA-treated and Tgfb1 +/− Col4a1 +/+ mice and n = 12, 8, and 7 for untreated, 4PBA-treated and Tgfb1 +/− Col4a1 + /Δex41 mice, respectively. Data are presented as mean ± SD. ** P < 0.01; **** P < 0.0001, unpaired two-tailed Student t -test for comparison between untreated Col4a1 +/+ and Col4a1 + /Δex41 mice. ‡ p < 0.05; ‡‡ p < 0.001, one-way ANOVA (or Kruskal-Wallis and Dunnett pairwise tests for non-Gaussian distribution in F and H) to test the effect of 4PBA treatment and Tgfb1 heterozygosity in mice of a given genotype. Additional parameters are shown in .

Article Snippet: The conditional Col4a1 Flex41 mutant mouse was generated by InGenious Targeting Laboratory (Stony Brook, NY) and previously characterized and validated [ , ].

Techniques: Generated, Two Tailed Test, Comparison

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Skeletal pathology in mouse models of Gould syndrome is partially alleviated by genetically reducing TGFβ signaling

doi: 10.1016/j.matbio.2024.07.005

Figure Lengend Snippet: 4PBA and Tgfb1 heterozygosity do not ameliorate alterations in bone mechanical and material properties in Col4a1 + /Δex41 mice. (A-F) 3-point bending flexural analysis of femurs from 3 to 4mo male mice showing altered femoral bone mechanical (A-C) and material (D-F) properties characterized by significant reductions in stiffness (A) and maximal load (C) and significant increase in elastic modulus (D) in Col4a1 + /Δex41 mice compared to Col4a1 +/+ mice. Neither 4PBA treatment nor Tgfb1 heterozygosity improved bone mechanical and material properties in Col4a1 + /Δex41 mice. 4PBA treatment did not affect bone mechanical and material properties in Col4a1 +/+ mice (A-F) but it exacerbated the reduction in femoral stiffness in Col4a1 + /Δex41 mice (A). While Tgfb1 heterozygosity had deleterious effects in Col4a1 +/+ mice, including significant reduction in maximal load (C) and increase in elastic modulus (D), it did not affect mechanical and material bone parameters in Col4a1 + /Δex41 mice. n = 9, 10, 8 for untreated, 4PBA-treated and Tgfb1 +/− Col4a1 +/+ mice, and n = 12, 8, and 7 for untreated, 4PBA-treated and Tgfb1 +/− Col4a1 + /Δex41 mice, respectively. Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; **** p < 0.0001, unpaired two-tailed Student t -test for comparison between untreated Col4a1 +/+ and Col4a1 + /Δex41 mice. ‡ p < 0.05; ‡‡ p < 0.001, one-way ANOVA (or Kruskal-Wallis and Dunnett pairwise tests for non-Gaussian distribution in A and F) to test the effect of 4PBA treatment and Tgfb1 heterozygosity in mice of a given genotype. Additional details are shown in .

Article Snippet: The conditional Col4a1 Flex41 mutant mouse was generated by InGenious Targeting Laboratory (Stony Brook, NY) and previously characterized and validated [ , ].

Techniques: Two Tailed Test, Comparison

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Skeletal pathology in mouse models of Gould syndrome is partially alleviated by genetically reducing TGFβ signaling

doi: 10.1016/j.matbio.2024.07.005

Figure Lengend Snippet: Reduced vascular density in femora from Col4a1 + /Δex41 mice. (A-B) Representative images and (C-E) quantification of synchrotron radiation micro-computed tomography (SRμCT) analysis of femoral longitudinal canals from 3 to 4 months old Col4a1 +/+ and Col4a1 + /Δex41 male mice. While vascular canal diameter was similar between Col4a1 +/+ and Col4a1 + /Δex41 mice (C), vascular canal density was significantly reduced in Col4a1 + /Δex41 mice (D) which was reflected in a trend towards decrease cortical porosity (E). Data are presented as mean ± SD. * p < 0.05.

Article Snippet: The conditional Col4a1 Flex41 mutant mouse was generated by InGenious Targeting Laboratory (Stony Brook, NY) and previously characterized and validated [ , ].

Techniques: Micro-CT

Journal: Circulation

Article Title: Molecular and Genetic Analysis of Collagen Type IV Mutant Mouse Models of Spontaneous Intracerebral Hemorrhage Identify Mechanisms for Stroke Prevention

doi: 10.1161/CIRCULATIONAHA.114.013395

Figure Lengend Snippet: Col4a1 mutation causes abnormal cerebral and retinal vascular development. (A) At embryonic day (E) 10.5 Col4a1+/Δex41 mice have irregularly shaped, enlarged blood vessels (calculated in branchial arches using CD31 and Collagen type IV (COL4) labeling, scale bar: 100 μm) and intracerebral hemorrhages (arrow). (B) At E12.5 Col4a1+/Δex41 mice have abnormally tortuous vessels with increased density (calculated in hindbrain flatmounts using CD31 labeling, scale bar: 100 μm) (C) In mutant animals, retinal blood vessels are tortuous, arteries (A) and veins (V) cross each other and there is excess branching in the main veins (5≤n≤16, pair-wise comparisons per age, scale bar: 500 μm). (D) Intracardiac perfusion of biotin revealed that adult mutant animals have tortuous cerebral blood vessels without compromised blood-brain-barrier (scale bar: 100 μm). Data are reported as mean + standard deviation and * indicates p<0.05 compared to Col4a1+/+ by Student’s t-test.

Article Snippet: The Col4a1 Flex41 conditional mutant mouse was produced by InGenious Targeting Laboratory (Stony Brook, NY).

Techniques: Mutagenesis, Labeling, Standard Deviation

Journal: Circulation

Article Title: Molecular and Genetic Analysis of Collagen Type IV Mutant Mouse Models of Spontaneous Intracerebral Hemorrhage Identify Mechanisms for Stroke Prevention

doi: 10.1161/CIRCULATIONAHA.114.013395

Figure Lengend Snippet: Col4a1 mutation causes small vessel disease, hemorrhagic stroke and macro-angiopathy. (A) Col4a1+/Δex41 mice have porencephaly (penetrance: 80%) and intracerebral hemorrhages (penetrance: 100%) quantified using Prussian blue staining of serial coronal brain sections (scale bar: 1 mm). Data are reported as mean ± standard deviation. (B) Macro-angiopathy with vessel wall thickening, fibrosis and thrombosis advanced with age. Trichrome staining shows collagen deposition (blue) and fibrin accumulation (red) in affected blood vessels (scale bar: 100 μm). No Col4a1+/+ mice had porencephaly, intracerebral hemorrhage or macro-angiopathy (n=32).

Article Snippet: The Col4a1 Flex41 conditional mutant mouse was produced by InGenious Targeting Laboratory (Stony Brook, NY).

Techniques: Mutagenesis, Staining, Standard Deviation

Journal: Circulation

Article Title: Molecular and Genetic Analysis of Collagen Type IV Mutant Mouse Models of Spontaneous Intracerebral Hemorrhage Identify Mechanisms for Stroke Prevention

doi: 10.1161/CIRCULATIONAHA.114.013395

Figure Lengend Snippet: Mutant Col4a1 causes cerebrovascular disease when expressed during vascular development. (A) Col4a1+/Flex41 conditional mutant mice were crossed to R26-CreER ubiquitous inducible CRE mice and the CRE recombinase was activated at different time points during or after vascular development by tamoxifen injections. (B) Quantification of retinal vessels revealed excess branching in mice that started to express mutant COL4A1 at birth but not at 1- or 3-weeks. n.s.: p>0.05, *: p<0.05 compared to Flex41 only (Col4a1+/Flex41; R26-CreER−/-) mice by Student’s t-test. (C) ICH quantification at 1 or 8 months of age shows that ICH severity increases with earlier age of expression of the collagen mutation. *: p<0.05 by Kruskal-Wallis test followed by Dunn’s post-test. Data are reported as mean ± standard deviation.

Article Snippet: The Col4a1 Flex41 conditional mutant mouse was produced by InGenious Targeting Laboratory (Stony Brook, NY).

Techniques: Mutagenesis, Expressing, Standard Deviation

Journal: Circulation

Article Title: Molecular and Genetic Analysis of Collagen Type IV Mutant Mouse Models of Spontaneous Intracerebral Hemorrhage Identify Mechanisms for Stroke Prevention

doi: 10.1161/CIRCULATIONAHA.114.013395

Figure Lengend Snippet: Environmental factors contribute to Col4a1 mutation expressivity. Intracerebral hemorrhage quantification in Col4a1+/Δex41 mice: (A) after vaginal delivery or Cesarean section, (B) without or with five sessions of five one-minute sprints on a treadmill, (C) without or with 7 days of Warfarin anticoagulant administration. Data are reported as mean ± standard deviation.

Article Snippet: The Col4a1 Flex41 conditional mutant mouse was produced by InGenious Targeting Laboratory (Stony Brook, NY).

Techniques: Mutagenesis, Standard Deviation

Journal: Circulation

Article Title: Molecular and Genetic Analysis of Collagen Type IV Mutant Mouse Models of Spontaneous Intracerebral Hemorrhage Identify Mechanisms for Stroke Prevention

doi: 10.1161/CIRCULATIONAHA.114.013395

Figure Lengend Snippet: Allelic heterogeneity and genetic context contribute to the expressivity of Col4a1 and Col4a2 mutations. (A) Intracerebral hemorrhage (ICH) quantification in 8-month-old mice with different mutations but the same genetic background, C57BL/6J (B6), demonstrates that allelic heterogeneity influences ICH severity. *: p<0.05 by Kruskal-Wallis test followed by Dunn’s post-test. (B) ICH quantification in 8-month-old mice with the same Col4a1Δex41 mutation but different genetic contexts shows that genetic background influences ICH severity. A cross for one generation to CAST/EiJ (CAST), but not 129S6/SvEvTac (129) mice was sufficient to reduce ICH severity compared to Col4a1+/Δex41 mice on a B6 background. *: p<0.05 by ANOVA followed by Tukey post-test. (C) CAST suppression of ICH was consistent across different mutations, however, (D) did not extend to prevention of vascular tortuosity and excess branching (COL4 labeling in the retina, CD31 labeling in the brain, scale bars: 50 μm). In (C) and (D) n.s. indicates p>0.05 and ** indicates p<0.01 by Student’s t-test. Data are reported as mean ± (A, B and C) or + (D) standard deviation.

Article Snippet: The Col4a1 Flex41 conditional mutant mouse was produced by InGenious Targeting Laboratory (Stony Brook, NY).

Techniques: Mutagenesis, Labeling, Standard Deviation

Journal: Circulation

Article Title: Molecular and Genetic Analysis of Collagen Type IV Mutant Mouse Models of Spontaneous Intracerebral Hemorrhage Identify Mechanisms for Stroke Prevention

doi: 10.1161/CIRCULATIONAHA.114.013395

Figure Lengend Snippet: Intracerebral hemorrhages associate with intracellular COL4A1 accumulation in vascular endothelial cells and pericytes but not with extracellular deficiency. (A) Immunolabeling and Western blot show that intracellular COL4A1 in Col4a1+/Δex41 cells from B6 mice is greatly reduced in cells from CASTB6F1 mice but without an obvious increase in extracellular COL4A1 (C: cell lysate, M: conditioned medium, Laminin and Tubulin are controls for secreted and intracellular proteins, respectively). Results constitute representative examples from twelve independent biological replicates obtained from three independent experiments. (B) Immunolabeling of Col4a1+/Δex41 retinal vessels shows strong intracellular collagen type IV (COL4) in B6 but not CASTB6F1 mice. (C) Col4a1+/Flex41 mice were crossed to Tie2-Cre, Pdgfrb-Cre or Gfap-Cre strains for mutant Col4a1 conditional expression in vascular endothelial cells (VECs), pericytes or astrocytes respectively. (D) Retinal vessel quantification revealed excess branching with mutant expression in VECs and pericytes but not astrocytes. n.s.: p>0.05, *: p<0.05 compared to Flex41-only mice (Col4a1+/Flex41; Cre−/-) by Student’s t-test. (E) Intracerebral hemorrhage (ICH) quantification demonstrated that mutant Col4a1 expression in VECs or pericytes is sufficient to cause ICH but that neither cell-type alone phenocopies Col4a1+/Δex41. Conditional expression of mutant Col4a1 in astrocytes caused very mild ICHs in only three 8-month-old animals. *: p<0.05 by Kruskal-Wallis test followed by Dunn’s post-test. Scale bars: 50 μm. Data are reported as mean + (D) or ± (E) standard deviation.

Article Snippet: The Col4a1 Flex41 conditional mutant mouse was produced by InGenious Targeting Laboratory (Stony Brook, NY).

Techniques: Immunolabeling, Western Blot, Mutagenesis, Expressing, Standard Deviation

Journal: Circulation

Article Title: Molecular and Genetic Analysis of Collagen Type IV Mutant Mouse Models of Spontaneous Intracerebral Hemorrhage Identify Mechanisms for Stroke Prevention

doi: 10.1161/CIRCULATIONAHA.114.013395

Figure Lengend Snippet: Treatment with chemical chaperone sodium 4-phenylbutyrate (4PBA) reduces intracerebral hemorrhage severity in vivo. Col4a1+/Δex41 mice treated from birth to 3 weeks with sodium 4-phenylbutyrate (4PBA) had similar levels of retinal vessel branching (A) but less severe intracerebral hemorrhages (ICHs) (B) compared to untreated Col4a1+/Δex41 mice. n.s.: p>0.05 by Student’s t-test. Immunolabeled vessels showed less intracellular and more extracellular collagen type IV (COL4) in the treated Col4a1+/Δex41 mouse with the least, compared to greatest, ICH severity. Scale bars: 50 μm. Data are reported as mean + (A) or ± (B) standard deviation.

Article Snippet: The Col4a1 Flex41 conditional mutant mouse was produced by InGenious Targeting Laboratory (Stony Brook, NY).

Techniques: In Vivo, Immunolabeling, Standard Deviation