Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Khan, Muznah; Gasser, Stephan

doi:10.3791/53565-v

Cited by 13 publications

(10 citation statements)

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“…Primary fibroblast cells from the ears of the G56S and WT mice were isolated using the protocol described by Khan and Gasser (26).…”

Section: Methodsmentioning

confidence: 99%

Impaired polyamine metabolism causes behavioral and neuroanatomical defects in a novel mouse model of Snyder-Robinson Syndrome

Kemaladewi

2023

Preprint

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Polyamines (putrescine, spermidine, and spermine) are essential for cellular growth and are subject to strict metabolic regulation. Mutations in the gene encoding spermine synthase (SMS) lead to the accumulation of spermidine and an X-linked recessive disorder known as Snyder-Robinson syndrome (SRS). There are no treatments available for this rare disease that manifests as mental retardation, thin habitus, and low muscle tone. The development of therapeutic interventions for SRS will require a suitable disease-specific animal model that recapitulates many of the abnormalities observed in these patients. Here, we characterize the molecular, behavioral, and neuroanatomical features of a mouse model with a missense mutation in Sms that results in a glycine-to-serine substitution at position 56 (G56S) of the SMS protein. Mice harboring this mutation exhibited a complete loss of SMS protein and elevated spermidine/spermine ratios in skeletal muscles and the brain. In addition, the G56S mice demonstrated increased anxiety, impaired learning, and decreased explorative behavior in fear conditioning, Morris water maze, and open field tests, respectively. Furthermore, the mice undergo significant reductions in body weight over time, as well as abnormalities in brain structure and bone density. Transcriptomic analysis of the cerebral cortex revealed downregulation of genes associated with mitochondrial oxidative phosphorylation and synthesis of ribosomal proteins. Our findings also revealed impaired mitochondrial bioenergetics in fibroblasts isolated from the G56S mice, which provides a link between these processes and the pathogenesis of SRS. Collectively, our findings establish the first in-depth characterization of SRS-associated mechanisms in a preclinical animal model and identify cellular processes that can be targeted for future therapeutic development.

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“…Primary fibroblast cells from the ears of the G56S and WT mice were isolated using the protocol described by Khan and Gasser (26).…”

Section: Methodsmentioning

confidence: 99%

Impaired polyamine metabolism causes behavioral and neuroanatomical defects in a novel mouse model of Snyder-Robinson Syndrome

Kemaladewi

2023

Preprint

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“…Murine dermal fibroblasts (MDFs) and skin explants were cultured as previously described (32). Briefly, mice were euthanized and shaved dorsal skin was excised and digested with collagenase D and pronase.…”

Section: Isolation Culture and Stimulation Of Cells And Tissuementioning

confidence: 99%

Loss of TRIM21 drives UVB-induced systemic inflammation by regulating DNA-sensing pathways

Tumurkhuu,

Moore,

Perri

et al. 2024

Preprint

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Background. Patients with systemic lupus erythematosus (SLE) experience photosensitivity, with exposure to ultraviolet light B (UVB) driving lupus flares and triggering symptoms like joint pain, fatigue, and cutaneous lesions. Although the mechanism(s) linking UVB exposure to systemic effects are unclear, type I interferons (IFNs) are known to play a role. Our previous work has shown that TRIM21, an autoantigen in SLE, functions as a negative regulator on the pathways driving IFN expression. Here we explore how TRIM21 functions to regulate both local and systemic inflammation following UVB exposure and that altered expression may drive cutaneous inflammation and photosensitivity in SLE. Methods. WT (C57BL/6) and Trim21-/- mice were irradiated with UVB (100mJ/cm2) on shaved dorsal region of the mice on consecutive days for 1 and 3 weeks, and UVB induced local cutaneous manifestations and systemic inflammation in blood, spleen and kidney were examined by messenger RNA expression of inflammatory and type I IFN response genes, histology, and flow cytometry. To determine the molecular targets of TRIM21 downstream of UVB, mechanistic studies were performed in bone marrow-derived macrophages (BMDMs) and mouse skin fibroblasts (MDF) from WT and Trim21-/- mice, and TRIM21-/- THP-1 cells. Results. Infiltration of inflammatory cells and induction of type I IFN developed in UVB-exposed areas in both sets of mice. Most notably after UVB exposure, we observed splenomegaly and enhanced expression of IFN-stimulated genes (ISG) in the blood and spleen of Trim21-/- mice. Subsequent analysis showed higher expression of Siglec1, an IFN-inducible protein, on Ly6Chi inflammatory monocytes in the spleen and blood cells of UVB-exposed Trim21-/- mice, indicating a systemic IFN response. Inflammatory chemokines CXCL10 and CXCL12, both important in UVB-induced skin inflammation, were also detected at significantly higher levels in serum of Trim21-/- mice after UVB exposure. In addition, Trim21-/- mice exposed to UVB also demonstrated enhanced total IgG levels in serum accompanied by increased skin deposition of IgG. Altogether, loss of Trim21 in mice results in enhanced systemic IFN-driven responses and mimics increased systemic disease in SLE patients following UVB exposure. TRIM21 acts to restrain RNA/DNA sensing and IFN responses, and enhanced basal and UVB- and cGAMP-dependent Ifnb1 expression was observed in Trim21-/- BMDMs and MDFs. In cells obtained from Trim21/Sting1 double knockout mice, cGAMP and cytoplasmic dsDNA induced Ifnb1 expression was reduced, indicating that TRIM21 regulates Ifnb1 expression through the adaptor protein STING. As TRIM21 is an E3 ligase and known to regulate protein stability, we assessed protein levels of cGAS, DDX41 and STING itself. Mechanistically, we found both degradation of DDX41 and STING levels were affected in stimulated Trim21-/- BMDMs. Taken together, our results indicate that TRIM21 protects against IFN induction at local and systemic levels due to a failure to restrict STING signaling. Understanding the role of TRIM21 in preventing systemic disease will have important understandings of its role in driving increased disease activity and flare in SLE.

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“…For time 0w, five mice were used. Tail skin from wound bed and distal memory areas (Distal) was dissected and processed as previously described 53 . After blocking, the following antibodies were incubated 10 min at RT at the concentration of 1,5 µg ml −1 : anti-mouse CD45 (VioGreen, Miltenyi Biotec 130-110-803), CD11b (clone M1/70) (FITC, Miltenyi Biotec 130-110-803), CD3 (clone 17.A2) (FITC, Miltenyi Biotec_130130-119-135), γδTCR (clone REA633) (PE-Vio770, Miltenyi Biotec_130-123-290), F4/80 (clone REA126) (PE-Vio770, Miltenyi Biotec 130-118-320), MHC-II (APC, Miltenyi Biotec 130-102-139) and CD206 (clone C068C2) (PE, BioLegend 141706).…”

Section: Flow Cytometry and Fluorescence-activated Cell Sortingmentioning

confidence: 99%

Tissue memory relies on stem cell priming in distal undamaged areas

et al. 2023

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Epithelial cells that participated in wound repair elicit a more efficient response to future injuries, which is believed to be locally restricted. Here we show that cell adaptation resulting from a localized tissue damage has a wide spatial impact at a scale not previously appreciated. We demonstrate that a specific stem cell population, distant from the original injury, originates long-lasting wound memory progenitors residing in their own niche. Notably, these distal memory cells have not taken part in the first healing but become intrinsically pre-activated through priming. This cell state, maintained at the chromatin and transcriptional level, leads to an enhanced wound repair that is partially recapitulated through epigenetic perturbation. Importantly wound memory has long-term harmful consequences, exacerbating tumourigenesis. Overall, we show that sub-organ-scale adaptation to injury relies on spatially organized memory-dedicated progenitors, characterized by an actionable cell state that establishes an epigenetic field cancerization and predisposes to tumour onset.

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Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Cited by 13 publications

References 0 publications

Impaired polyamine metabolism causes behavioral and neuroanatomical defects in a novel mouse model of Snyder-Robinson Syndrome

Impaired polyamine metabolism causes behavioral and neuroanatomical defects in a novel mouse model of Snyder-Robinson Syndrome

Loss of TRIM21 drives UVB-induced systemic inflammation by regulating DNA-sensing pathways

Tissue memory relies on stem cell priming in distal undamaged areas

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