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1.
Proc Natl Acad Sci U S A ; 115(46): E10907-E10914, 2018 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-30381459

RESUMEN

The translational potential of cell-based therapies is often limited by complications related to effectively engineering and manufacturing functional cells. While the use of electroporation is widespread, the impact of electroporation on cell state and function has yet to be fully characterized. Here, we use a genome-wide approach to study optimized electroporation treatment and identify striking disruptions in the expression profiles of key functional transcripts of human T cells. These genetic disruptions result in concomitant perturbation of cytokine secretion including a 648-fold increase in IL-2 secretion (P < 0.01) and a 30-fold increase in IFN-γ secretion (P < 0.05). Ultimately, the effects at the transcript and protein level resulted in functional deficiencies in vivo, with electroporated T cells failing to demonstrate sustained antigen-specific effector responses when subjected to immunological challenge. In contrast, cells subjected to a mechanical membrane disruption-based delivery mechanism, cell squeezing, had minimal aberrant transcriptional responses [0% of filtered genes misregulated, false discovery rate (FDR) q < 0.1] relative to electroporation (17% of genes misregulated, FDR q < 0.1) and showed undiminished effector responses, homing capabilities, and therapeutic potential in vivo. In a direct comparison of functionality, T cells edited for PD-1 via electroporation failed to distinguish from untreated controls in a therapeutic tumor model, while T cells edited with similar efficiency via cell squeezing demonstrated the expected tumor-killing advantage. This work demonstrates that the delivery mechanism used to insert biomolecules affects functionality and warrants further study.


Asunto(s)
Ingeniería Celular/métodos , Microfluídica/métodos , Células Dendríticas/inmunología , Electroporación/métodos , Humanos , ARN Mensajero/metabolismo , Linfocitos T/inmunología , Transcriptoma
2.
Dev Biol ; 433(2): 461-472, 2018 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-29111100

RESUMEN

Activation of progenitor cells is crucial to promote tissue repair following injury in adult animals. In the context of successful limb regeneration following amputation, progenitor cells residing within the stump must re-enter the cell cycle to promote regrowth of the missing limb. We demonstrate that in axolotls, amputation is sufficient to induce cell-cycle activation in both the amputated limb and the intact, uninjured contralateral limb. Activated cells were found throughout all major tissue populations of the intact contralateral limb, with internal cellular populations (bone and soft tissue) the most affected. Further, activated cells were additionally found within the heart, liver, and spinal cord, suggesting that amputation induces a common global activation signal throughout the body. Among two other injury models, limb crush and skin excisional wound, only limb crush injuries were capable of inducing cellular responses in contralateral uninjured limbs but did not achieve activation levels seen following limb loss. We found this systemic activation response to injury is independent of formation of a wound epidermis over the amputation plane, suggesting that injury-induced signals alone can promote cellular activation. In mammals, mTOR signaling has been shown to promote activation of quiescent cells following injury, and we confirmed a subset of activated contralateral cells is positive for mTOR signaling within axolotl limbs. These findings suggest that conservation of an early systemic response to injury exists between mammals and axolotls, and propose that a distinguishing feature in species capable of full regeneration is converting this initial activation into sustained and productive growth at the site of regeneration.


Asunto(s)
Ambystoma mexicanum/fisiología , Ciclo Celular , Regeneración/fisiología , Ambystoma mexicanum/lesiones , Amputación Quirúrgica , Animales , Regeneración Ósea/fisiología , Cartílago/fisiología , Lesiones por Aplastamiento/fisiopatología , Replicación del ADN , Dermis/fisiología , Epidermis/fisiología , Extremidades/lesiones , Extremidades/fisiología , Regeneración Nerviosa/fisiología , Especificidad de Órganos , Células Satélite del Músculo Esquelético/fisiología , Piel/lesiones , Serina-Treonina Quinasas TOR/fisiología , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología
3.
Hum Mol Genet ; 24(2): 436-49, 2015 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-25209981

RESUMEN

Harlequin ichthyosis (HI) is a severe skin disease which leads to neonatal death in ∼50% of cases. It is the result of mutations in ABCA12, a protein that transports lipids required to establish the protective skin barrier needed after birth. To better understand the life-threatening newborn HI phenotype, we analysed the developing epidermis for consequences of lipid dysregulation in mouse models. We observed a pro-inflammatory signature which was characterized by chemokine upregulation in embryonic skin which is distinct from that seen in other types of ichthyosis. Inflammation also persisted in grafted HI skin. To examine the contribution of inflammation to disease development, we overexpressed interleukin-37b to globally suppress fetal inflammation, observing considerable improvements in keratinocyte differentiation. These studies highlight inflammation as an unexpected contributor to HI disease development in utero, and suggest that inhibiting inflammation may reduce disease severity.


Asunto(s)
Ictiosis Lamelar/embriología , Ictiosis Lamelar/inmunología , Animales , Diferenciación Celular , Quimiocinas/genética , Quimiocinas/inmunología , Modelos Animales de Enfermedad , Epidermis/embriología , Epidermis/inmunología , Femenino , Humanos , Ictiosis Lamelar/genética , Ictiosis Lamelar/fisiopatología , Interleucina-1/genética , Interleucina-1/inmunología , Queratinocitos/citología , Masculino , Ratones , Ratones Noqueados , Fenotipo , Piel/embriología , Piel/inmunología
4.
PLoS Genet ; 10(10): e1004706, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25340345

RESUMEN

Keratins are cytoskeletal intermediate filament proteins that are increasingly being recognised for their diverse cellular functions. Here we report the consequences of germ line inactivation of Keratin 76 (Krt76) in mice. Homozygous disruption of this epidermally expressed gene causes neonatal skin flaking, hyperpigmentation, inflammation, impaired wound healing, and death prior to 12 weeks of age. We show that this phenotype is associated with functionally defective tight junctions that are characterised by mislocalization of the integral protein CLDN1. We further demonstrate that KRT76 interacts with CLDN1 and propose that this interaction is necessary to correctly position CLDN1 in tight junctions. The mislocalization of CLDN1 has been associated in various dermopathies, including the inflammatory disease, psoriasis. These observations establish a previously unknown connection between the intermediate filament cytoskeleton network and tight junctions and showcase Krt76 null mice as a possible model to study aberrant tight junction driven skin diseases.


Asunto(s)
Claudina-1/genética , Queratinas/genética , Psoriasis/genética , Enfermedades de la Piel/genética , Uniones Estrechas/genética , Animales , Citoesqueleto/genética , Epidermis/metabolismo , Epidermis/patología , Humanos , Filamentos Intermedios/genética , Filamentos Intermedios/patología , Queratinocitos/metabolismo , Ratones , Psoriasis/patología , Enfermedades de la Piel/patología , Uniones Estrechas/patología
5.
PLoS Genet ; 10(10): e1004705, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25340873

RESUMEN

The skin is a highly regenerative organ which plays critical roles in protecting the body and sensing its environment. Consequently, morbidity and mortality associated with skin defects represent a significant health issue. To identify genes important in skin development and homeostasis, we have applied a high throughput, multi-parameter phenotype screen to the conditional targeted mutant mice generated by the Wellcome Trust Sanger Institute's Mouse Genetics Project (Sanger-MGP). A total of 562 different mouse lines were subjected to a variety of tests assessing cutaneous expression, macroscopic clinical disease, histological change, hair follicle cycling, and aberrant marker expression. Cutaneous lesions were associated with mutations in 23 different genes. Many of these were not previously associated with skin disease in the organ (Mysm1, Vangl1, Trpc4ap, Nom1, Sparc, Farp2, and Prkab1), while others were ascribed new cutaneous functions on the basis of the screening approach (Krt76, Lrig1, Myo5a, Nsun2, and Nf1). The integration of these skin specific screening protocols into the Sanger-MGP primary phenotyping pipelines marks the largest reported reverse genetic screen undertaken in any organ and defines approaches to maximise the productivity of future projects of this nature, while flagging genes for further characterisation.


Asunto(s)
Mutación/genética , Fenotipo , Fenómenos Fisiológicos de la Piel/genética , Animales , Células Madre Embrionarias , Folículo Piloso/metabolismo , Folículo Piloso/fisiología , Ratones , Genética Inversa
6.
Proc Natl Acad Sci U S A ; 110(8): 2928-33, 2013 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-23382243

RESUMEN

The spindle assembly checkpoint (SAC) ensures correct chromosome segregation during mitosis by preventing aneuploidy, an event that is detrimental to the fitness and survival of normal cells but oncogenic in tumor cells. Deletion of SAC genes is incompatible with early mouse development, and RNAi-mediated depletion of SAC components in cultured cells results in rapid death. Here we describe the use of a conditional KO of mouse Mad2, an essential component of the SAC signaling cascade, as a means to selectively induce chromosome instability and aneuploidy in the epidermis of the skin. We observe that SAC inactivation is tolerated by interfollicular epidermal cells but results in depletion of hair follicle bulge stem cells. Eventually, a histologically normal epidermis develops within ∼1 mo after birth, albeit without any hair. Mad2-deficient cells in this epidermis exhibited abnormal transcription of metabolic genes, consistent with aneuploid cell state. Hair follicle bulge stem cells were completely absent, despite the continued presence of rudimentary hair follicles. These data demonstrate that different cell lineages within a single tissue respond differently to chromosome instability: some proliferating cell lineages can survive, but stem cells are highly sensitive.


Asunto(s)
Células Epidérmicas , Cabello/citología , Huso Acromático , Células Madre/citología , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Citometría de Flujo , Inmunohistoquímica , Hibridación Fluorescente in Situ , Proteínas Mad2 , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Interferencia de ARN
7.
bioRxiv ; 2024 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-39257748

RESUMEN

Previous studies have reported that amputation invokes body-wide responses in regenerative organisms, but most have not examined the implications of these changes beyond the region of tissue regrowth. Specifically, long-range epidermal responses to amputation are largely uncharacterized, with research on amputation-induced epidermal responses in regenerative organisms traditionally being restricted to the wound site. Here, we investigate the effect of amputation on long-range epidermal permeability in two evolutionarily distant, regenerative organisms: axolotls and planarians. We find that amputation triggers a long-range increase in epidermal permeability in axolotls, accompanied by a long-range epidermal downregulation in MAPK signaling. Additionally, we provide functional evidence that pharmacologically inhibiting MAPK signaling in regenerating planarians increases long-range epidermal permeability. These findings advance our knowledge of body-wide changes due to amputation in regenerative organisms and warrant further study on whether epidermal permeability dysregulation in the context of amputation may lead to pathology in both regenerative and non-regenerative organisms.

8.
Nat Commun ; 13(1): 2833, 2022 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-35595757

RESUMEN

The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Endonucleasas/genética , Endonucleasas/metabolismo , Edición Génica/métodos , Células HEK293 , Humanos , ARN/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo
9.
Birth Defects Res C Embryo Today ; 90(1): 8-31, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20301220

RESUMEN

Basement membranes (BMs) are specializations of the extracellular matrix that act as key mediators of development and disease. Their sheet like protein matrices typically serve to separate epithelial or endothelial cell layers from underlying mesenchymal tissues, providing both a biophysical support to overlying tissue as well as a hub to promote and regulate cell-cell and cell-protein interactions. In the latter context, the BM is increasingly being recognized as a mediator of growth factor interactions during development. In this review, we discuss recent findings regarding the structure of the BM and its roles in mediating the normal development of the embryo, and we examine congenital diseases affecting the BM which impact embryonic development and health in later life.


Asunto(s)
Membrana Basal/embriología , Anomalías Congénitas/embriología , Desarrollo Embrionario/fisiología , Matriz Extracelular/fisiología , Animales , Membrana Basal/patología , Membrana Basal/fisiología , Anomalías Congénitas/patología , Anomalías Congénitas/fisiopatología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/fisiología , Proteoglicanos de Heparán Sulfato/fisiología , Humanos
10.
J Cell Biol ; 216(1): 247-263, 2017 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-27998989

RESUMEN

Human ciliopathies, including Joubert syndrome (JBTS), arise from cilia dysfunction. The inositol polyphosphate 5-phosphatase INPP5E localizes to cilia and is mutated in JBTS. Murine Inpp5e ablation is embryonically lethal and recapitulates JBTS, including neural tube defects and polydactyly; however, the underlying defects in cilia signaling and the function of INPP5E at cilia are still emerging. We report Inpp5e-/- embryos exhibit aberrant Hedgehog-dependent patterning with reduced Hedgehog signaling. Using mouse genetics, we show increasing Hedgehog signaling via Smoothened M2 expression rescues some Inpp5e-/- ciliopathy phenotypes and "normalizes" Hedgehog signaling. INPP5E's phosphoinositide substrates PI(4,5)P2 and PI(3,4,5)P3 accumulated at the transition zone (TZ) in Hedgehog-stimulated Inpp5e-/- cells, which was associated with reduced recruitment of TZ scaffolding proteins and reduced Smoothened levels at cilia. Expression of wild-type, but not 5-phosphatase-dead, INPP5E restored TZ molecular organization and Smoothened accumulation at cilia. Therefore, we identify INPP5E as an essential point of convergence between Hedgehog and phosphoinositide signaling at cilia that maintains TZ function and Hedgehog-dependent embryonic development.


Asunto(s)
Anomalías Múltiples/enzimología , Cerebelo/anomalías , Cilios/enzimología , Embrión de Mamíferos/enzimología , Anomalías del Ojo/enzimología , Enfermedades Renales Quísticas/enzimología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Retina/anomalías , Epitelio Pigmentado de la Retina/enzimología , Sistemas de Mensajero Secundario , Anomalías Múltiples/genética , Animales , Línea Celular , Cerebelo/enzimología , Modelos Animales de Enfermedad , Anomalías del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Predisposición Genética a la Enfermedad , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Enfermedades Renales Quísticas/genética , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/genética , Retina/enzimología , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Factores de Tiempo , Transfección , Proteína Gli2 con Dedos de Zinc
11.
Cell Rep ; 18(3): 762-776, 2017 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-28099853

RESUMEN

Mammals have extremely limited regenerative capabilities; however, axolotls are profoundly regenerative and can replace entire limbs. The mechanisms underlying limb regeneration remain poorly understood, partly because the enormous and incompletely sequenced genomes of axolotls have hindered the study of genes facilitating regeneration. We assembled and annotated a de novo transcriptome using RNA-sequencing profiles for a broad spectrum of tissues that is estimated to have near-complete sequence information for 88% of axolotl genes. We devised expression analyses that identified the axolotl orthologs of cirbp and kazald1 as highly expressed and enriched in blastemas. Using morpholino anti-sense oligonucleotides, we find evidence that cirbp plays a cytoprotective role during limb regeneration whereas manipulation of kazald1 expression disrupts regeneration. Our transcriptome and annotation resources greatly complement previous transcriptomic studies and will be a valuable resource for future research in regenerative biology.


Asunto(s)
Extremidades/fisiología , Transcriptoma , Ambystoma mexicanum , Animales , Hibridación in Situ , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/antagonistas & inhibidores , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , ARN/química , ARN/metabolismo , Interferencia de ARN , Empalme del ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Regeneración , Análisis de Secuencia de ARN
12.
Regeneration (Oxf) ; 2(1): 37-43, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27499866

RESUMEN

Loss-of-function genetics provides strong evidence for a gene's function in a wild-type context. In many model systems, this approach has been invaluable for discovering the function of genes in diverse biological processes. Axolotls are urodele amphibians (salamanders) with astonishing regenerative abilities, capable of regenerating entire limbs, portions of the tail (including spinal cord), heart, and brain into adulthood. With their relatively short generation time among salamanders, they offer an outstanding opportunity to interrogate natural mechanisms for appendage and organ regeneration provided that the tools are developed to address these long-standing questions. Here we demonstrate targeted modification of the thrombospondin-1 (tsp-1) locus using transcription-activator-like effector nucleases (TALENs) and identify a role of tsp-1 in recruitment of myeloid cells during limb regeneration. We find that while tsp-1-edited mosaic animals still regenerate limbs, they exhibit a reduced subepidermal collagen layer in limbs and an increased number of myeloid cells within blastemas. This work presents a protocol for generating and genotyping mosaic axolotls with TALEN-mediated gene edits.

13.
Nat Commun ; 5: 3540, 2014 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-24721909

RESUMEN

Permanent stop-and-shop large-scale mouse mutant resources provide an excellent platform to decipher tissue phenogenomics. Here we analyse skin from 538 knockout mouse mutants generated by the Sanger Institute Mouse Genetics Project. We optimize immunolabelling of tail epidermal wholemounts to allow systematic annotation of hair follicle, sebaceous gland and interfollicular epidermal abnormalities using ontology terms from the Mammalian Phenotype Ontology. Of the 50 mutants with an epidermal phenotype, 9 map to human genetic conditions with skin abnormalities. Some mutant genes are expressed in the skin, whereas others are not, indicating systemic effects. One phenotype is affected by diet and several are incompletely penetrant. In-depth analysis of three mutants, Krt76, Myo5a (a model of human Griscelli syndrome) and Mysm1, provides validation of the screen. Our study is the first large-scale genome-wide tissue phenotype screen from the International Knockout Mouse Consortium and provides an open access resource for the scientific community.


Asunto(s)
Fenotipo , Genética Inversa/métodos , Piel , Animales , Bases de Datos Genéticas , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Mutación
14.
Cell Metab ; 18(2): 225-38, 2013 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-23931754

RESUMEN

ABCA12 is involved in the transport of ceramides in skin, but it may play a wider role in lipid metabolism. We show that, in Abca12-deficient macrophages, cholesterol efflux failed to respond to activation with LXR agonists. Abca12 deficiency caused a reduction in the abundance of Abca1, Abcg1, and Lxrß. Overexpression of Lxrß reversed the effects. Mechanistically, Abca12 deficiency did not affect expression of genes involved in cholesterol metabolism. Instead, a physical association between Abca1, Abca12, and Lxrß proteins was established. Abca12 deficiency enhanced interaction between Abca1 and Lxrß and the degradation of Abca1. Overexpression of ABCA12 in HeLa-ABCA1 cells increased the abundance and stability of ABCA1. Abca12 deficiency caused an accumulation of cholesterol in macrophages and the formation of foam cells, impaired reverse cholesterol transport in vivo, and increased the development of atherosclerosis in irradiated Apoe(-/-) mice reconstituted with Apoe(-/-)Abca12(-/-) bone marrow. Thus, ABCA12 regulates the cellular cholesterol metabolism via an LXRß-dependent posttranscriptional mechanism.


Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Colesterol/metabolismo , Macrófagos/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/deficiencia , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Transporte Biológico/genética , Línea Celular , Células Espumosas/metabolismo , Células HeLa , Humanos , Metabolismo de los Lípidos/genética , Lipoproteínas/metabolismo , Receptores X del Hígado , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Nucleares Huérfanos/biosíntesis
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