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1.
Prostaglandins Other Lipid Mediat ; 169: 106769, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37625781

RESUMO

Charcot-Marie-Tooth Disease (CMT) is a commonly inherited peripheral polyneuropathy. Clinical manifestations for this disease include symmetrical distal polyneuropathy, altered deep tendon reflexes, distal sensory loss, foot deformities, and gait abnormalities. Genetic mutations in heat shock proteins have been linked to CMT2. Specifically, mutations in the heat shock protein B1 (HSPB1) gene encoding for heat shock protein 27 (Hsp27) have been linked to CMT2F and distal hereditary motor and sensory neuropathy type 2B (dHMSN2B) subtype. The goal of the study was to examine the role of an endogenous mutation in HSPB1 in vivo and to define the effects of this mutation on motor function and pathology in a novel animal model. As sphingolipids have been implicated in hereditary and sensory neuropathies, we examined sphingolipid metabolism in central and peripheral nervous tissues in 3-month-old HspS139F mice. Though sphingolipid levels were not altered in sciatic nerves from HspS139F mice, ceramides and deoxyceramides, as well as sphingomyelins (SMs) were elevated in brain tissues from HspS139F mice. Histology was utilized to further characterize HspS139F mice. HspS139F mice exhibited no alterations to the expression and phosphorylation of neurofilaments, or in the expression of acetylated α-tubulin in the brain or sciatic nerve. Interestingly, HspS139F mice demonstrated cerebellar demyelination. Locomotor function, grip strength and gait were examined to define the role of HspS139F in the clinical phenotypes associated with CMT2F. Gait analysis revealed no differences between HspWT and HspS139F mice. However, both coordination and grip strength were decreased in 3-month-old HspS139F mice. Together these data suggest that the endogenous S139F mutation in HSPB1 may serve as a mouse model for hereditary and sensory neuropathies such as CMT2F.


Assuntos
Doença de Charcot-Marie-Tooth , Camundongos , Animais , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Proteínas de Choque Térmico/genética , Mutação/genética , Modelos Animais de Doenças , Esfingolipídeos
2.
J Immunol ; 205(7): 1763-1777, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32868408

RESUMO

The CD27-CD70 costimulatory pathway is essential for the full activation of T cells, but some studies show that blocking this pathway exacerbates certain autoimmune disorders. In this study, we report on the impact of CD27-CD70 signaling on disease progression in the NOD mouse model of type 1 diabetes (T1D). Specifically, our data demonstrate that CD70 ablation alters thymocyte selection and increases circulating T cell levels. CD27 signaling was particularly important for the thymic development and peripheral homeostasis of Foxp3+Helios+ regulatory T cells, which likely accounts for our finding that CD70-deficient NOD mice develop more-aggressive T1D onset. Interestingly, we found that CD27 signaling suppresses the thymic development and effector functions of T1D-protective invariant NKT cells. Thus, rather than providing costimulatory signals, the CD27-CD70 axis may represent a coinhibitory pathway for this immunoregulatory T cell population. Moreover, we showed that a CD27 agonist Ab reversed the effects of CD70 ablation, indicating that the phenotypes observed in CD70-deficient mice were likely due to a lack of CD27 signaling. Collectively, our results demonstrate that the CD27-CD70 costimulatory pathway regulates the differentiation program of multiple T cell subsets involved in T1D development and may be subject to therapeutic targeting.


Assuntos
Ligante CD27/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Células T Matadoras Naturais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Ligante CD27/genética , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunomodulação , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Transdução de Sinais , Fatores de Transcrição/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
3.
Mamm Genome ; 32(6): 415-426, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34477920

RESUMO

Rhomboid proteases, first discovered in Drosophila, are intramembrane serine proteases. Members of the rhomboid protein family that are catalytically deficient are known as inactive rhomboids (iRhoms). iRhoms have been implicated in wound healing, cancer, and neurological disorders such as Alzheimer's and Parkinson's diseases, inflammation, and skin diseases. The past decade of mouse research has shed new light on two key protein domains of iRhoms-the cytosolic N-terminal domain and the transmembrane dormant peptidase domain-suggesting new ways to target multiple intracellular signaling pathways. This review focuses on recent advances in uncovering the unique functions of iRhom protein domains in normal growth and development, growth factor signaling, and inflammation, with a perspective on future therapeutic opportunities.


Assuntos
Neoplasias , Serina Proteases , Animais , Modelos Animais de Doenças , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Serina Proteases/metabolismo , Transdução de Sinais
4.
FASEB J ; 34(11): 15252-15268, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32959379

RESUMO

Sphingolipids have been implicated in mammalian placental development and function, but their regulation in the placenta remains unclear. Herein we report that alkaline ceramidase 2 (ACER2) plays a key role in sustaining the integrity of the placental vasculature by regulating the homeostasis of sphingolipids in mice. The mouse alkaline ceramidase 2 gene (Acer2) is highly expressed in the placenta between embryonic day (E) 9.5 and E12.5. Acer2 deficiency in both the mother and fetus decreases the placental levels of sphingolipids, including sphingoid bases (sphingosine and dihydrosphingosine) and sphingoid base-1-phosphates (sphingosine-1-phosphate and dihydrosphingosine-1-phosphate) and results in the in utero death of ≈50% of embryos at E12.5 whereas Acer2 deficiency in either the mother or fetus has no such effects. Acer2 deficiency causes hemorrhages from the maternal vasculature in the junctional and/or labyrinthine zones in E12.5 placentas. Moreover, hemorrhagic but not non-hemorrhagic Acer2-deficient placentas exhibit an expansion of parietal trophoblast giant cells with a concomitant decrease in the area of the fetal blood vessel network in the labyrinthine zone, suggesting that Acer2 deficiency results in embryonic lethality due to the atrophy of the fetal blood vessel network in the placenta. Taken together, these results suggest that ACER2 sustains the integrity of the placental vasculature by controlling the homeostasis of sphingolipids in mice.


Assuntos
Ceramidase Alcalina/fisiologia , Hemorragia/patologia , Lisofosfolipídeos/metabolismo , Placenta/patologia , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Doenças Vasculares/patologia , Animais , Feminino , Hemorragia/etiologia , Hemorragia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Placenta/metabolismo , Gravidez , Esfingosina/metabolismo , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismo
5.
BMC Genomics ; 21(1): 856, 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-33267773

RESUMO

BACKGROUND: The development and application of CRISPR technologies for the modification of the genome are rapidly expanding. Advances in the field describe new CRISPR components that are strategically engineered to improve the precision and reliability of CRISPR editing within the genome sequence. Genome modification using induced genome breaks that are targeted and mediated by CRISPR components leverage cellular mechanisms for repair like homology directed repair (HDR) to incorporate genomic edits with increased precision. RESULTS: In this report, we describe the gain of methylation at typically hypomethylated CpG island (CGI) locations affected by the CRISPR-mediated incorporation of donor DNA using HDR mechanisms. With characterization of CpG methylation patterns using whole genome bisulfite sequencing, these CGI methylation disruptions trace the insertion of the donor DNA during the genomic edit. These insertions mediated by homology-directed recombination disrupt the generational methylation pattern stability of the edited CGI within the cells and their cellular lineage within the animal strain, persisting across generations. Our approach describes a statistically based workflow for indicating locations of modified CGIs and provides a mechanism for evaluating the directed modification of the methylome of the affected CGI at the CpG-level. CONCLUSIONS: With advances in genome modification technology comes the need to detect the level and persistence of methylation change that modifications to the genomic sequence impose upon the collaterally edited methylome. Any modification of the methylome of somatic or germline cells could have implications for gene regulation mechanisms governed by the methylation patterns of CGI regions in the application of therapeutic edits of more sensitively regulated genomic regions. The method described here locates the directed modification of the mouse epigenome that persists over generations. While this observance would require supporting molecular observations such as direct sequence changes or gene expression changes, the observation of epigenetic modification provides an indicator that intentionally directed genomic edits can lead to collateral, unintentional epigenomic changes post modification with generational persistence.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genoma , Animais , Ilhas de CpG/genética , Metilação de DNA , Epigenoma , Edição de Genes , Camundongos , Reprodutibilidade dos Testes
6.
FASEB J ; 33(3): 3137-3151, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30383447

RESUMO

Immunodeficient mice engrafted with human peripheral blood mononuclear cells (PBMCs) support preclinical studies of human pathogens, allograft rejection, and human T-cell function. However, a major limitation of PBMC engraftment is development of acute xenogeneic graft- versus-host disease (GVHD) due to human T-cell recognition of murine major histocompatibility complex (MHC). To address this, we created 2 NOD- scid IL-2 receptor subunit γ ( IL2rg) null (NSG) strains that lack murine MHC class I and II [NSG-ß-2-microglobulin ( B2M) null ( IA IE)null and NSG -( Kb Db) null ( IAnull)]. We observed rapid human IgG clearance in NSG- B2Mnull ( IA IE) null mice whereas clearance in NSG -( Kb Db) null ( IAnull) mice and NSG mice was comparable. Injection of human PBMCs into both strains enabled long-term engraftment of human CD4+ and CD8+ T cells without acute GVHD. Engrafted human T-cell function was documented by rejection of human islet allografts. Administration of human IL-2 to NSG -( Kb Db) null ( IAnull) mice via adeno-associated virus vector increased human CD45+ cell engraftment, including an increase in human regulatory T cells. However, high IL-2 levels also induced the development of GVHD. These data document that NSG mice deficient in murine MHC support studies of human immunity in the absence of acute GVHD and enable evaluation of human antibody therapeutics targeting human T cells.-Brehm, M. A., Kenney, L. L., Wiles, M. V., Low, B. E., Tisch, R. M., Burzenski, L., Mueller, C., Greiner, D. L., Shultz, L. D. Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.


Assuntos
Doença Enxerto-Hospedeiro/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/transplante , Linfócitos T/imunologia , Animais , Feminino , Genes MHC Classe I , Genes MHC da Classe II , Sobrevivência de Enxerto/imunologia , Xenoenxertos , Humanos , Transplante das Ilhotas Pancreáticas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Fenótipo
7.
J Am Soc Nephrol ; 30(12): 2322-2336, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31558682

RESUMO

BACKGROUND: Lithium, mainstay treatment for bipolar disorder, causes nephrogenic diabetes insipidus and hypercalcemia in about 20% and 10% of patients, respectively, and may lead to acidosis. These adverse effects develop in only a subset of patients treated with lithium, suggesting genetic factors play a role. METHODS: To identify susceptibility genes for lithium-induced adverse effects, we performed a genome-wide association study in mice, which develop such effects faster than humans. On day 8 and 10 after assigning female mice from 29 different inbred strains to normal chow or lithium diet (40 mmol/kg), we housed the animals for 48 hours in metabolic cages for urine collection. We also collected blood samples. RESULTS: In 17 strains, lithium treatment significantly elevated urine production, whereas the other 12 strains were not affected. Increased urine production strongly correlated with lower urine osmolality and elevated water intake. Lithium caused acidosis only in one mouse strain, whereas hypercalcemia was found in four strains. Lithium effects on blood pH or ionized calcium did not correlate with effects on urine production. Using genome-wide association analyses, we identified eight gene-containing loci, including a locus containing Acer2, which encodes a ceramidase and is specifically expressed in the collecting duct. Knockout of Acer2 led to increased susceptibility for lithium-induced diabetes insipidus development. CONCLUSIONS: We demonstrate that genome-wide association studies in mice can be used successfully to identify susceptibility genes for development of lithium-induced adverse effects. We identified Acer2 as a first susceptibility gene for lithium-induced diabetes insipidus in mice.


Assuntos
Ceramidase Alcalina/genética , Diabetes Insípido Nefrogênico/genética , Cloreto de Lítio/toxicidade , Equilíbrio Ácido-Base/fisiologia , Acidose/induzido quimicamente , Acidose/genética , Animais , Diabetes Insípido Nefrogênico/induzido quimicamente , Dinoprostona/urina , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Hematócrito , Hipercalcemia/induzido quimicamente , Hipercalcemia/genética , Túbulos Renais Coletores/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Néfrons/metabolismo , RNA Mensageiro/biossíntese , Sódio/sangue , Especificidade da Espécie
8.
FASEB J ; 32(6): 3058-3069, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29401619

RESUMO

Sphingosine-1-phosphate (S1P) plays important roles in cardiovascular development and immunity. S1P is abundant in plasma because erythrocytes-the major source of S1P-lack any S1P-degrading activity; however, much remains unclear about the source of the plasma S1P precursor, sphingosine (SPH), derived mainly from the hydrolysis of ceramides by the action of ceramidases that are encoded by 5 distinct genes, acid ceramidase 1 ( ASAH1)/ Asah1, ASAH2/ Asah2, alkaline ceramidase 1 ( ACER1)/ Acer1, ACER2/ Acer2, and ACER3/ Acer3, in humans/mice. Previous studies have reported that knocking out Asah1 or Asah2 failed to reduce plasma SPH and S1P levels in mice. In this study, we show that knocking out Acer1 or Acer3 also failed to reduce the blood levels of SPH or S1P in mice. In contrast, knocking out Acer2 from either whole-body or the hematopoietic lineage markedly decreased the blood levels of SPH and S1P in mice. Of interest, knocking out Acer2 from whole-body or the hematopoietic lineage also markedly decreased the levels of dihydrosphingosine (dhSPH) and dihydrosphingosine-1-phosphate (dhS1P) in blood. Taken together, these results suggest that ACER2 plays a key role in the maintenance of high plasma levels of sphingoid base-1-phosphates-S1P and dhS1P-by controlling the generation of sphingoid bases-SPH and dhSPH-in hematopoietic cells.-Li, F., Xu, R., Low, B. E., Lin, C.-L., Garcia-Barros, M., Schrandt, J., Mileva, I., Snider, A., Luo, C. K., Jiang, X.-C., Li, M.-S., Hannun, Y. A., Obeid, L. M., Wiles, M. V., Mao, C. Alkaline ceramidase 2 is essential for the homeostasis of plasma sphingoid bases and their phosphates.


Assuntos
Ceramidase Alcalina/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Hemostasia/fisiologia , Lisofosfolipídeos/sangue , Esfingolipídeos/sangue , Esfingosina/análogos & derivados , Esfingosina/sangue , Ceramidase Alcalina/genética , Animais , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Knockout
9.
J Cell Biochem ; 118(10): 3043-3048, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28332231

RESUMO

Mice are the most commonly used model organisms to study human disease. Many genetic human diseases can be recapitulated by modifying the mouse genome allowing the testing of existing and novel therapeutics, including combinatorial therapeutics, without putting humans at risk. Specifically, the development of "humanized" mice, that is, severely immunodeficient mice engrafted with functional human hematopoietic and immune cells and tissues, has revolutionized our ability to study and model human diseases in preclinical in vivo systems. Until recently it has been challenging to develop strains of humanized mice with targeted mutations or that transgenically express human genes with site-specific mutations, and can permit optimal growth of functional human cells and tissues. However, recent advances in targeted nuclease-based genetic engineering have enabled precise modification and development of humanized mouse models at an unprecedented pace. These modifications permit optimal growth of functional human cells and tissues and can be used to replicate human genetically determined diseases. J. Cell. Biochem. 118: 3043-3048, 2017. © 2017 Wiley Periodicals, Inc.


Assuntos
Modelos Animais de Doenças , Edição de Genes , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Animais , Doenças Genéticas Inatas/terapia , Humanos , Camundongos , Camundongos Transgênicos
10.
Exp Mol Pathol ; 102(2): 337-346, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28268192

RESUMO

Rhomboid family protein RHBDF2, an upstream regulator of the epidermal growth factor (EGF) receptor signaling, has been implicated in cutaneous wound healing. However, the underlying molecular mechanisms are still emerging. In humans, a gain-of-function mutation in the RHBDF2 gene accelerates cutaneous wound healing in an EGFR-dependent manner. Likewise, a gain-of-function mutation in the mouse Rhbdf2 gene (Rhbdf2cub/cub) shows a regenerative phenotype (rapid ear-hole closure) resulting from constitutive activation of the EGFR pathway. Because the RHBDF2-regulated EGFR pathway is relevant to cutaneous wound healing in humans, we used Rhbdf2cub/cub mice to investigate the biological networks and pathways leading to accelerated ear-hole closure, with the goal of identifying therapeutic targets potentially effective in promoting wound healing in humans. Comparative transcriptome analysis of ear pinna tissue from Rhbdf2cub/cub and Rhbdf2+/+ mice at 0h, 15min, 2h, and 24h post-wounding revealed an early induction of the nuclear factor E2-related factor 2 (NRF2)-mediated anti-oxidative pathway (0h and 15min), followed by the integrin-receptor aggregation pathway (2h) as early-stage events immediately and shortly after wounding in Rhbdf2cub/cub mice. Additionally, we observed genes enriched for the Fc fragment of the IgG receptor IIIa (FCGR3A)-mediated phagocytosis pathway 24h post-wounding. Although cutaneous wound repair in healthy individuals is generally non-problematic, it can be severely impaired due to aging, diabetes, and chronic inflammation. This study suggests that activation of the NRF2-antioxidant pathway by rhomboid protein RHBDF2 might be beneficial in treating chronic non-healing wounds.


Assuntos
Antioxidantes , Proteínas de Transporte/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Cicatrização , Animais , Proteínas de Transporte/genética , Modelos Animais de Doenças , Orelha/lesões , Receptores ErbB/genética , Receptores ErbB/metabolismo , Perfilação da Expressão Gênica , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Fenótipo , Fosforilação , Receptores de IgG/genética , Receptores de IgG/metabolismo , Regeneração , Transdução de Sinais
11.
Mamm Genome ; 26(9-10): 501-10, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25991564

RESUMO

CRISPR and CRISPR-associated (Cas) proteins, which in nature comprise the RNA-based adaptive immune system in bacteria and archaea, have emerged as particularly powerful genome editing tools owing to their unrivaled ease of use and ability to modify genomes across mammalian model systems. As such, the CRISPR-Cas9 system holds promise as a "system of choice" for functional mammalian genetic studies across biological disciplines. Here we briefly review this fast moving field, introduce the CRISPR-Cas9 system and its application to genome editing, with a focus on the basic considerations in designing the targeting guide RNA sequence.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de RNA/genética , RNA Guia de Cinetoplastídeos/genética , Animais , Sequência de Bases/genética , Engenharia Genética , Genoma
12.
J Investig Dermatol Symp Proc ; 17(2): 27-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26551941

RESUMO

Technology now exists for rapid screening of mutated laboratory mice to identify phenotypes associated with specific genetic mutations. Large repositories exist for spontaneous mutants and those induced by chemical mutagenesis, many of which have never been fully studied or comprehensively evaluated. To supplement these resources, a variety of techniques have been consolidated in an international effort to create mutations in all known protein coding genes in the mouse. With targeted embryonic stem cell lines now available for almost all protein coding genes and more recently CRISPR/Cas9 technology, large-scale efforts are underway to create further novel mutant mouse strains and to characterize their phenotypes. However, accurate diagnosis of skin, hair, and nail diseases still relies on careful gross and histological analysis, and while not automated to the level of the physiological phenotyping, histopathology still provides the most direct and accurate diagnosis and correlation with human diseases. As a result of these efforts, many new mouse dermatological disease models are being characterized and developed.


Assuntos
Alopecia em Áreas/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Animais , Genoma , Humanos , Camundongos , Camundongos Knockout , Fenótipo
13.
bioRxiv ; 2024 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-37292807

RESUMO

Inhibitory G alpha (GNAI or Gαi) proteins are critical for the polarized morphogenesis of sensory hair cells and for hearing. The extent and nature of their actual contributions remains unclear, however, as previous studies did not investigate all GNAI proteins and included non-physiological approaches. Pertussis toxin can downregulate functionally redundant GNAI1, GNAI2, GNAI3 and GNAO proteins, but may also induce unrelated defects. Here we directly and systematically determine the role(s) of each individual GNAI protein in mouse auditory hair cells. GNAI2 and GNAI3 are similarly polarized at the hair cell apex with their binding partner GPSM2, whereas GNAI1 and GNAO are not detected. In Gnai3 mutants, GNAI2 progressively fails to fully occupy the subcellular compartments where GNAI3 is missing. In contrast, GNAI3 can fully compensate for the loss of GNAI2 and is essential for hair bundle morphogenesis and auditory function. Simultaneous inactivation of Gnai2 and Gnai3 recapitulates for the first time two distinct types of defects only observed so far with pertussis toxin: 1) a delay or failure of the basal body to migrate off-center in prospective hair cells, and 2) a reversal in the orientation of some hair cell types. We conclude that GNAI proteins are critical for hair cells to break planar symmetry and to orient properly before GNAI2/3 regulate hair bundle morphogenesis with GPSM2.

14.
Elife ; 122024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38651641

RESUMO

Inhibitory G alpha (GNAI or Gαi) proteins are critical for the polarized morphogenesis of sensory hair cells and for hearing. The extent and nature of their actual contributions remains unclear, however, as previous studies did not investigate all GNAI proteins and included non-physiological approaches. Pertussis toxin can downregulate functionally redundant GNAI1, GNAI2, GNAI3, and GNAO proteins, but may also induce unrelated defects. Here, we directly and systematically determine the role(s) of each individual GNAI protein in mouse auditory hair cells. GNAI2 and GNAI3 are similarly polarized at the hair cell apex with their binding partner G protein signaling modulator 2 (GPSM2), whereas GNAI1 and GNAO are not detected. In Gnai3 mutants, GNAI2 progressively fails to fully occupy the sub-cellular compartments where GNAI3 is missing. In contrast, GNAI3 can fully compensate for the loss of GNAI2 and is essential for hair bundle morphogenesis and auditory function. Simultaneous inactivation of Gnai2 and Gnai3 recapitulates for the first time two distinct types of defects only observed so far with pertussis toxin: (1) a delay or failure of the basal body to migrate off-center in prospective hair cells, and (2) a reversal in the orientation of some hair cell types. We conclude that GNAI proteins are critical for hair cells to break planar symmetry and to orient properly before GNAI2/3 regulate hair bundle morphogenesis with GPSM2.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Células Ciliadas Auditivas , Morfogênese , Animais , Camundongos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/fisiologia , Polaridade Celular , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética
15.
PLoS One ; 18(10): e0292456, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37796769

RESUMO

Previous work strongly implicated Collagen 17a1 (Col17a1) as a potent genetic modifier of junctional epidermolysis bullosa (JEB) caused by a hypomorphic mutation (Lamc2jeb) in mice. The importance of the noncollagenous domain (NC4) of COLXVII was suggested by use of a congenic reduction approach that restricted the modifier effect to 2-3 neighboring amino acid changes in that domain. The current study utilizes TALEN and CRISPR/Cas9 induced amino acid replacements and in-frame indels nested to NC4 to further investigate the role of this and adjoining COLXVII domains both as modifiers and primary risk effectors. We confirm the importance of COLXVI AA 1275 S/G and 1277 N/S substitutions and utilize small nested indels to show that subtle changes in this microdomain attenuate JEB. We further show that large in-frame indels removing up to 1482 bp and 169 AA of NC6 through NC1 domains are surprisingly disease free on their own but can be very potent modifiers of Lamc2jeb/jeb JEB. Together these studies exploiting gene editing to functionally dissect the Col17a1 modifier demonstrate the importance of epistatic interactions between a primary disease-causing mutation in one gene and innocuous 'healthy' alleles in other genes.


Assuntos
Epidermólise Bolhosa Juncional , Animais , Camundongos , Epidermólise Bolhosa Juncional/genética , Colágenos não Fibrilares/genética , Colágenos não Fibrilares/metabolismo , Colágeno/genética , Mutação , Aminoácidos/genética
16.
PLoS One ; 18(10): e0293218, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37883475

RESUMO

The Lamc2jeb junctional epidermolysis bullosa (EB) mouse model has been used to demonstrate that significant genetic modification of EB symptoms is possible, identifying as modifiers Col17a1 and six other quantitative trait loci, several with strong candidate genes including dystonin (Dst/Bpag1). Here, CRISPR/Cas9 was used to alter exon 23 in mouse skin specific isoform Dst-e (Ensembl GRCm38 transcript name Dst-213, transcript ID ENSMUST00000183302.5, protein size 2639AA) and validate a proposed arginine/glutamine difference at amino acid p1226 in B6 versus 129 mice as a modifier of EB. Frame shift deletions (FSD) in mouse Dst-e exon 23 (Dst-eFSD/FSD) were also identified that cause mice carrying wild-type Lamc2 to develop a phenotype similar to human EB simplex without dystonia musculorum. When combined, Dst-eFSD/FSD modifies Lamc2jeb/jeb (FSD+jeb) induced disease in unexpected ways implicating an altered balance between DST-e (BPAG1e) and a rarely reported rodless DST-eS (BPAG1eS) in epithelium as a possible mechanism. Further, FSD+jeb mice with pinnae removed are found to provide a test bed for studying internal epithelium EB disease and treatment without severe skin disease as a limiting factor while also revealing and accelerating significant nasopharynx symptoms present but not previously noted in Lamc2jeb/jeb mice.


Assuntos
Distonia , Distúrbios Distônicos , Epidermólise Bolhosa Simples , Epidermólise Bolhosa Juncional , Epidermólise Bolhosa , Animais , Camundongos , Distonia/genética , Distonia/metabolismo , Distúrbios Distônicos/metabolismo , Distonina/metabolismo , Epidermólise Bolhosa/genética , Epidermólise Bolhosa Simples/diagnóstico , Epidermólise Bolhosa Simples/genética , Epidermólise Bolhosa Simples/metabolismo , Epidermólise Bolhosa Juncional/genética , Epidermólise Bolhosa Juncional/diagnóstico , Epidermólise Bolhosa Juncional/metabolismo , Pele/metabolismo
17.
Front Bioeng Biotechnol ; 10: 910151, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35866031

RESUMO

The inability to insert large DNA constructs into the genome efficiently and precisely is a key challenge in genomic engineering. Random transgenesis, which is widely used, lacks precision, and comes with a slew of drawbacks. Lentiviral and adeno-associated viral methods are plagued by, respectively, DNA toxicity and a payload capacity of less than 5 kb. Homology-directed repair (HDR) techniques based on CRISPR-Cas9 can be effective, but only in the 1-5 kb range. In addition, long homology arms-DNA sequences that permit construct insertion-of lengths ranging from 0.5 to 5 kb are required by currently known HDR-based techniques. A potential new method that uses Cas9-guided transposases to insert DNA structures up to 10 kb in length works well in bacteria, but only in bacteria. Surmounting these roadblocks, a new toolkit has recently been developed that combines RNA-guided Cas9 and the site-specific integrase Bxb1 to integrate DNA constructs ranging in length from 5 to 43 kb into mouse zygotes with germline transmission and into human cells. This ground-breaking toolkit will give researchers a valuable resource for developing novel, urgently needed mouse and human induced pluripotent stem cell (hiPSC) models of cancer and other genetic diseases, as well as therapeutic gene integration and biopharmaceutical applications, such as the development of stable cell lines to produce therapeutic protein products.

18.
Sci Rep ; 12(1): 5424, 2022 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-35361849

RESUMO

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene "landing pad" composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies > 40% with up to ~ 43 kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~ 4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.


Assuntos
Bacteriófagos , COVID-19 , Animais , Bacteriófagos/genética , DNA , Técnicas de Transferência de Genes , Patrimônio Genético , Integrases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos NOD , RNA Viral , SARS-CoV-2
19.
Dis Model Mech ; 15(9)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36037073

RESUMO

The lack of genetically diverse preclinical animal models in basic biology and efficacy testing has been cited as a potential cause of failure in clinical trials. We developed and characterized five diverse RAG1 null mouse strains as models that allow xenografts to grow. In these strains, we characterized the growth of breast cancer, leukemia and glioma cell lines. We found a wide range of growth characteristics that were far more dependent on strain than tumor type. For the breast cancer cell line, we characterized the spectrum of xenograft/tumor growth at structural, histological, cellular and molecular levels across each strain, and found that each strain captures unique structural components of the stroma. Furthermore, we showed that the increase in tumor-infiltrating myeloid CD45+ cells and the amount of circulating cytokine IL-6 and chemokine KC (also known as CXCL1) is associated with a higher tumor size in different strains. This resource is available to study established human xenografts, as well as difficult-to-xenograft tumors and growth of hematopoietic stems cells, and to decipher the role of myeloid cells in the development of spontaneous cancers.


Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Knockout , Transplante Heterólogo , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Cells ; 10(9)2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34571853

RESUMO

The dysregulation of microRNA (miRNA) is implicated in cancer, inflammation, cardiovascular disorders, drug resistance, and aging. While most researchers study miRNA's role as a biomarker, for example, to distinguish between various sub-forms or stages of a given disease of interest, research is also ongoing to utilize these small nucleic acids as therapeutics. An example of a common pleiotropic disease that could benefit from miRNA-based therapeutics is inflammatory bowel disease (IBD), which is characterized by chronic inflammation of the small and large intestines. Due to complex interactions between multiple factors in the etiology of IBD, development of therapies that effectively maintain remission for this disease is a significant challenge. In this review, we discuss the role of dysregulated miRNA expression in the context of clinical ulcerative colitis (UC) and Crohn's disease (CD)-the two main forms of IBD-and the various preclinical mouse models of IBD utilized to validate the therapeutic potential of targeting these miRNA. Additionally, we highlight advances in the development of genetically engineered animal models that recapitulate clinical miRNA expression and provide powerful preclinical models to assess the diagnostic and therapeutic promise of miRNA in IBD.


Assuntos
Doenças Inflamatórias Intestinais/genética , MicroRNAs/genética , Animais , Biomarcadores , Colite Ulcerativa/genética , Doença de Crohn/genética , Modelos Animais de Doenças , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Doenças Inflamatórias Intestinais/imunologia , Camundongos , MicroRNAs/fisiologia
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