RESUMO
Posttranslational modification of a protein with glycosylphosphatidylinositol (GPI) is a conserved mechanism exists in all eukaryotes. Thus far, >150 human GPI-anchored proteins have been discovered and ~30 enzymes have been reported to be involved in the biosynthesis and maturation of mammalian GPI. Phosphatidylinositol glycan biosynthesis class A protein (PIGA) catalyzes the very first step of GPI anchor biosynthesis. Patients carrying a mutation of the PIGA gene usually suffer from inherited glycosylphosphatidylinositol deficiency (IGD) with intractable epilepsy and intellectual developmental disorder. We generated three mouse models with PIGA deficits specifically in telencephalon excitatory neurons (Ex-M-cko), inhibitory neurons (In-M-cko) or thalamic neurons (Th-H-cko), respectively. Both Ex-M-cko and In-M-cko mice showed impaired long-term fear memory and were more susceptible to kainic acid-induced seizures. In addition, In-M-cko demonstrated a severe limb-clasping phenotype. Hippocampal synapse changes were observed in Ex-M-cko mice. Our Piga conditional knockout mouse models provide powerful tools to understand the cell-type specific mechanisms underlying inherited GPI deficiency and to test different therapeutic modalities.
Assuntos
Glicosilfosfatidilinositóis , Ácido Caínico , Animais , Cognição , Glicosilfosfatidilinositóis/deficiência , Humanos , Ácido Caínico/metabolismo , Mamíferos , Camundongos , Camundongos Knockout , Mutação , Neurônios/metabolismo , Convulsões/genética , Convulsões/metabolismoRESUMO
Interferon-γ (IFN-γ) is essential for host defense against intracellular pathogens. Stimulation of innate immune cells by IFN-γ upregulates â¼2,000 effector genes such as immunity-related GTPases including p65 guanylate-binding protein (Gbp) family genes. We show that a cluster of Gbp genes was required for host cellular immunity against the intracellular parasite Toxoplasma gondii. We generated mice deficient for all six Gbp genes located on chromosome 3 (Gbp(chr3)) by targeted chromosome engineering. Mice lacking Gbp(chr3) were highly susceptible to T. gondii infection, resulting in increased parasite burden in immune organs. Furthermore, Gbp(chr3)-deleted macrophages were defective in IFN-γ-mediated suppression of T. gondii intracellular growth and recruitment of IFN-γ-inducible p47 GTPase Irgb6 to the parasitophorous vacuole. In addition, some members of Gbp(chr3) restored the protective response against T. gondii in Gbp(chr3)-deleted cells. Our results suggest that Gbp(chr3) play a pivotal role in anti-T. gondii host defense by controlling IFN-γ-mediated Irgb6-dependent cellular innate immunity.
Assuntos
Proteínas de Ligação ao GTP/imunologia , Imunidade Inata/imunologia , Interferon gama/imunologia , Macrófagos/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Cromossomos de Mamíferos/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Imunidade Celular/imunologia , Interferon gama/antagonistas & inibidores , Interferon gama/metabolismo , Listeria monocytogenes/imunologia , Medições Luminescentes , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Óxido Nítrico/metabolismo , Toxoplasma/metabolismoRESUMO
Haploid mouse embryonic stem cells (ESCs), in which a single hit mutation is sufficient to produce loss-of-function phenotypes, have provided a powerful tool for forward genetic screening. This strategy, however, can be hampered by undesired autodiploidization of haploid ESCs. To overcome this obstacle, we designed a new methodology that facilitates enrichment of homozygous mutant ESC clones arising from autodiploidization during haploid gene trap mutagenesis. Haploid mouse ESCs were purified by fluorescence-activated cell sorting to maintain their haploid property and then transfected with the Tol2 transposon-based biallelically polyA-trapping (BPATrap) vector that carries an invertible G418 plus puromycin double selection cassette. G418 plus puromycin double selection enriched biallelic mutant clones that had undergone autodiploidization following a single vector insertion into the haploid genome. Using this method, we successfully generated 222 homozygous mutant ESCs from 2208 clones by excluding heterozygous ESCs and ESCs with multiple vector insertions. This relatively low efficiency of generating homozygous mutant ESCs was partially overcome by cell sorting of haploid ESCs after Tol2 BPATrap transfection. These results demonstrate the feasibility of our approach to provide an efficient platform for mutagenesis of ESCs and functional analysis of the mammalian genome.
Assuntos
Homozigoto , Células-Tronco Embrionárias Murinas/fisiologia , Mutagênese/genética , Animais , Células Cultivadas , Elementos de DNA Transponíveis , Diploide , Citometria de Fluxo , Vetores Genéticos , Gentamicinas/farmacologia , Haploidia , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Poli A , Puromicina/farmacologia , Reprodutibilidade dos TestesRESUMO
BACKGROUND: A delicate balance between cell death and keratinocyte proliferation is crucial for normal skin development. Previous studies have reported that cellular FLICE (FADD-like ICE)-inhibitory protein plays a crucial role in prevention of keratinocytes from TNF-α-dependent apoptosis and blocking of dermatitis. However, a role for cellular FLICE-inhibitory protein in TNF-α-independent cell death remains unclear. OBJECTIVE: We investigated contribution of TNF-α-dependent and TNF-α-independent signals to the development of dermatitis in epidermis-specific Cflar-deficient (CflarE-KO) mice. METHODS: We examined the histology and expression of epidermal differentiation markers and inflammatory cytokines in the skin of CflarE-KO;Tnfrsf1a+/- and CflarE-KO;Tnfrsf1a-/- mice. Mice were treated with neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand to block TNF-α-independent cell death of CflarE-KO;Tnfrsf1a-/- mice. RESULTS: CflarE-KO;Tnfrsf1a-/- mice were born but experienced severe dermatitis and succumbed soon after birth. CflarE-KO;Tnfrsf1a+/- mice exhibited embryonic lethality caused by massive keratinocyte apoptosis. Although keratinocytes from CflarE-KO;Tnfrsf1a-/- mice still died of apoptosis, neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand substantially prolonged survival of CflarE-KO;Tnfrsf1a-/- mice. Expression of inflammatory cytokines, such as Il6 and Il17a was increased; conversely, expression of epidermal differentiation markers was severely downregulated in the skin of CflarE-KO;Tnfrsf1a-/- mice. Treatment of primary keratinocytes with IL-6 and, to a lesser extent, IL-17A suppressed expression of epidermal differentiation markers. CONCLUSION: TNF receptor superfamily 1 (TNFR1)-dependent or TNFR1-independent apoptosis of keratinocytes promotes inflammatory cytokine production, which subsequently blocks epidermal differentiation. Thus blockade of both TNFR1-dependent and TNFR1-independent cell death might be an alternative strategy to treat skin diseases when treatment with anti-TNF-α antibody alone is not sufficient.
Assuntos
Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Dermatite/imunologia , Epiderme/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Apoptose/genética , Apoptose/imunologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Dermatite/genética , Dermatite/patologia , Epiderme/patologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Camundongos , Camundongos Knockout , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/imunologiaRESUMO
Protein quality control in cardiomyocytes is crucial to maintain cellular homeostasis. The accumulation of damaged organelles, such as mitochondria and misfolded proteins in the heart is associated with heart failure. During the process to identify novel mitochondria-specific autophagy (mitophagy) receptors, we found FK506-binding protein 8 (FKBP8), also known as FKBP38, shares similar structural characteristics with a yeast mitophagy receptor, autophagy-related 32 protein. However, knockdown of FKBP8 had no effect on mitophagy in HEK293 cells or H9c2 myocytes. Since the role of FKBP8 in the heart has not been fully elucidated, the aim of this study is to determine the functional role of FKBP8 in the heart. Cardiac-specific FKBP8-deficient (Fkbp8-/-) mice were generated. Fkbp8-/- mice showed no cardiac phenotypes under baseline conditions. The Fkbp8-/- and control wild type littermates (Fkbp8+/+) mice were subjected to pressure overload by means of transverse aortic constriction (TAC). Fkbp8-/- mice showed left ventricular dysfunction and chamber dilatation with lung congestion 1week after TAC. The number of apoptotic cardiomyocytes was dramatically elevated in TAC-operated Fkbp8-/- hearts, accompanied with an increase in protein levels of cleaved caspase-12 and endoplasmic reticulum (ER) stress markers. Caspase-12 inhibition resulted in the attenuation of hydrogen peroxide-induced apoptotic cell death in FKBP8 knockdown H9c2 myocytes. Immunocytological and immunoprecipitation analyses indicate that FKBP8 is localized to the ER and mitochondria in the isolated cardiomyocytes, interacting with heat shock protein 90. Furthermore, there was accumulation of misfolded protein aggregates in FKBP8 knockdown H9c2 myocytes and electron dense deposits in perinuclear region in TAC-operated Fkbp8-/- hearts. The data suggest that FKBP8 plays a protective role against hemodynamic stress in the heart mediated via inhibition of the accumulation of misfolded proteins and ER-associated apoptosis.
Assuntos
Apoptose , Cardiotônicos/metabolismo , Retículo Endoplasmático/metabolismo , Coração/fisiopatologia , Hemodinâmica , Estresse Fisiológico , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Aorta/patologia , Apoptose/efeitos dos fármacos , Caspase 12/metabolismo , Constrição Patológica , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Coração/efeitos dos fármacos , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Hemodinâmica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/toxicidade , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mitofagia/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Especificidade de Órgãos , Pressão , Ligação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Ratos Sprague-Dawley , Transdução de Sinais , Estresse Fisiológico/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Ligação a Tacrolimo/deficiência , Remodelação Ventricular/efeitos dos fármacosRESUMO
BACKGROUND: CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive. RESULTS: Here, we show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats. CONCLUSIONS: The high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice.
Assuntos
Engenharia Genética/métodos , Alelos , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Injeções , Camundongos , Camundongos Knockout , Zigoto/metabolismoRESUMO
An important challenge in cancer genomics is precise detection of structural variations (SVs) by high-throughput short-read sequencing, which is hampered by the high false discovery rates of existing analysis tools. Here, we propose an accurate SV detection method named COSMOS, which compares the statistics of the mapped read pairs in tumor samples with isogenic normal control samples in a distinct asymmetric manner. COSMOS also prioritizes the candidate SVs using strand-specific read-depth information. Performance tests on modeled tumor genomes revealed that COSMOS outperformed existing methods in terms of F-measure. We also applied COSMOS to an experimental mouse cell-based model, in which SVs were induced by genome engineering and gamma-ray irradiation, followed by polymerase chain reaction-based confirmation. The precision of COSMOS was 84.5%, while the next best existing method was 70.4%. Moreover, the sensitivity of COSMOS was the highest, indicating that COSMOS has great potential for cancer genome analysis.
Assuntos
Biologia Computacional/métodos , Células-Tronco Embrionárias/citologia , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Análise de Sequência de DNA/métodos , Animais , Variação Genética/genética , Genoma Humano/genética , Humanos , CamundongosRESUMO
From November 17 to 20 in 2015, the Conference on Transposition and Genome Engineering 2015 (TGE 2015) was held at Nara Kasugano International Forum-IRAKA-in Nara, Japan, located at the center of Nara Park. All of the presentations were carried out at Nohgaku hall in Nara Kasugano International Forum-IRAKA. Participation totaled 148 persons (30 international, 118 domestic), who were able to engage in lively scientific discussions over the 4-day period. The guest speaker list consisted of many top-notch international researchers, an achievement for which the conference received praise from the attendees. There were 36 oral presentations including the keynote lecture (22 presentations from guest speakers, complemented with 14 selected from abstract submissions). Additionally, there were 46 poster presentations. The conference uniquely combined research mainly from two different genomics approaches: (i) transposon technology allowing random genomic integration followed by gene discovery-related phenotypes and (ii) genome editing technology with designer nuclease allowing precise modification of a gene-of-interest.
Assuntos
Elementos de DNA Transponíveis , Engenharia Genética/métodos , Genômica/métodos , JapãoRESUMO
Bloom syndrome, an autosomal recessive disorder of the BLM gene, confers predisposition to a broad spectrum of early-onset cancers in multiple tissue types. Loss of genomic integrity is a primary hallmark of such human malignancies, but many studies using disease-affected specimens are limited in that they are retrospective and devoid of an appropriate experimental control. To overcome this, we devised an experimental system to recapitulate the early molecular events in genetically engineered mouse embryonic stem cells, in which cells undergoing loss of heterozygosity (LOH) can be enriched after inducible down-regulation of Blm expression, with or without site-directed DNA double-strand break (DSB) induction. Transient loss of BLM increased the rate of LOH, whose breakpoints were distributed along the chromosome. Combined with site-directed DSB induction, loss of BLM synergistically increased the rate of LOH and concentrated the breakpoints around the targeted chromosomal region. We characterized the LOH events using specifically tailored genomic tools, such as high-resolution array comparative genomic hybridization and high-density single nucleotide polymorphism genotyping, revealing that the combination of BLM suppression and DSB induction enhanced genomic rearrangements, including deletions and insertions, whose breakpoints were clustered in genomic inverted repeats and associated with junctional microhomologies. Our experimental approach successfully uncovered the detailed molecular mechanisms of as-yet-uncharacterized loss of heterozygosities and reveals the significant contribution of microhomology-mediated genomic rearrangements, which could be widely applicable to the early steps of cancer formation in general.
Assuntos
Síndrome de Bloom/genética , Instabilidade Genômica , Recombinação Homóloga , RecQ Helicases/genética , Animais , Linhagem Celular , Aberrações Cromossômicas , Pontos de Quebra do Cromossomo , Quebras de DNA de Cadeia Dupla , Regulação para Baixo , Células-Tronco Embrionárias/metabolismo , Conversão Gênica , Heterozigoto , Camundongos , Polimorfismo de Nucleotídeo Único , RecQ Helicases/metabolismoRESUMO
Although the roles of acids in bone metabolism are well characterized, the function of proton-sensing receptors in bone metabolism remains to be explored. In this study, we evaluated the role of proton-sensing receptor T-cell death-associated gene 8 (TDAG8) in osteoclastic activity during bone loss after ovariectomy. Through observations of bone mineral content, we found that pathological bone resorption was significantly exacerbated in mice homozygous for a gene trap mutation in the Tdag8 gene. Furthermore, osteoclasts from the homozygous mutant mice resorbed calcium in vitro more than the osteoclasts from the heterozygous mice did. Impaired osteoclast formation under acidic conditions was ameliorated in cultures of bone marrow cells by Tdag8 gene mutation. Extracellular acidification changed the cell morphology of osteoclasts via the TDAG8-Rho signaling pathway. These results suggest that the enhancement of TDAG8 function represents a new strategy for preventing bone resorption diseases, such as osteoporosis.
Assuntos
Reabsorção Óssea/metabolismo , Osteoclastos/metabolismo , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Microscopia Confocal , Ovariectomia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb(-/-)). Rheb(-/-) mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb(-/-) was lower than that in the control (Rheb(+/+)) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb(+/+) mice but not in Rheb(-/-) mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb(-/-) hearts during the neonatal period. Rheb(-/-) hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb(-/-) hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb(-/-) mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/crescimento & desenvolvimento , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neuropeptídeos/metabolismo , Serina-Treonina Quinases TOR/química , Proteínas Adaptadoras de Transdução de Sinal , Animais , Animais Recém-Nascidos , Autofagia , Southern Blotting , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Proliferação de Células , Cromossomos Artificiais Bacterianos , Ecocardiografia/métodos , Fatores de Iniciação em Eucariotos , Coração/fisiologia , Hipertrofia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Modelos Genéticos , Células Musculares/citologia , Miocárdio/metabolismo , Fosfoproteínas/metabolismo , Polirribossomos/metabolismo , Biossíntese de Proteínas , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Transdução de Sinais , Fatores de TempoRESUMO
BACKGROUND: Genome-wide saturation mutagenesis and subsequent phenotype-driven screening has been central to a comprehensive understanding of complex biological processes in classical model organisms such as flies, nematodes, and plants. The degree of "saturation" (i.e., the fraction of possible target genes identified) has been shown to be a critical parameter in determining all relevant genes involved in a biological function, without prior knowledge of their products. In mammalian model systems, however, the relatively large scale and labor intensity of experiments have hampered the achievement of actual saturation mutagenesis, especially for recessive traits that require biallelic mutations to manifest detectable phenotypes. RESULTS: By exploiting the recently established haploid mouse embryonic stem cells (ESCs), we present an implementation of almost complete saturation mutagenesis in a mammalian system. The haploid ESCs were mutagenized with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and processed for the screening of mutants defective in various steps of the glycosylphosphatidylinositol-anchor biosynthetic pathway. The resulting 114 independent mutant clones were characterized by a functional complementation assay, and were shown to be defective in any of 20 genes among all 22 known genes essential for this well-characterized pathway. Ten mutants were further validated by whole-exome sequencing. The predominant generation of single-nucleotide substitutions by ENU resulted in a gene mutation rate proportional to the length of the coding sequence, which facilitated the experimental design of saturation mutagenesis screening with the aid of computational simulation. CONCLUSIONS: Our study enables mammalian saturation mutagenesis to become a realistic proposition. Computational simulation, combined with a pilot mutagenesis experiment, could serve as a tool for the estimation of the number of genes essential for biological processes such as drug target pathways when a positive selection of mutants is available.
Assuntos
Células-Tronco Embrionárias/efeitos dos fármacos , Haploidia , Mutagênese/efeitos dos fármacos , Mutação/efeitos dos fármacos , Animais , Simulação por Computador , Células-Tronco Embrionárias/metabolismo , Etilnitrosoureia/farmacologia , Genoma , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Camundongos , Mutagênese/genética , Mutação/genética , FenótipoRESUMO
Microphthalmia with limb anomalies (MLA) is a rare autosomal-recessive disorder, presenting with anophthalmia or microphthalmia and hand and/or foot malformation. We mapped the MLA locus to 14q24 and successfully identified three homozygous (one nonsense and two splice site) mutations in the SPARC (secreted protein acidic and rich in cysteine)-related modular calcium binding 1 (SMOC1) in three families. Smoc1 is expressed in the developing optic stalk, ventral optic cup, and limbs of mouse embryos. Smoc1 null mice recapitulated MLA phenotypes, including aplasia or hypoplasia of optic nerves, hypoplastic fibula and bowed tibia, and syndactyly in limbs. A thinned and irregular ganglion cell layer and atrophy of the anteroventral part of the retina were also observed. Soft tissue syndactyly, resulting from inhibited apoptosis, was related to disturbed expression of genes involved in BMP signaling in the interdigital mesenchyme. Our findings indicate that SMOC1/Smoc1 is essential for ocular and limb development in both humans and mice.
Assuntos
Deformidades Congênitas dos Membros/genética , Microftalmia/genética , Osteonectina/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 14/genética , Códon sem Sentido/genética , Extremidades/crescimento & desenvolvimento , Olho/crescimento & desenvolvimento , Genes Recessivos , Loci Gênicos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Nervo Óptico/anormalidades , Splicing de RNA/genética , Síndrome de Waardenburg/genéticaRESUMO
Genome-wide mutagenesis in mouse embryonic stem cells (ESCs) is a powerful tool, but the diploid nature of the mammalian genome hampers its application for recessive genetic screening. We have previously reported a method to induce homozygous mutant ESCs from heterozygous mutants by tetracycline-dependent transient disruption of the Bloom's syndrome gene. However, we could not purify homozygous mutants from a large population of heterozygous mutant cells, limiting the applications. Here we developed a strategy for rapid enrichment of homozygous mutant mouse ESCs and demonstrated its feasibility for cell-based phenotypic analysis. The method uses G418-plus-puromycin double selection to enrich for homozygotes and single-nucleotide polymorphism analysis for identification of homozygosity. We combined this simple approach with gene-trap mutagenesis to construct a homozygous mutant ESC bank with 138 mutant lines and demonstrate its use in phenotype-driven genetic screening.
Assuntos
Bancos de Espécimes Biológicos , Células-Tronco Embrionárias/metabolismo , Homozigoto , Mutação/genética , Bancos de Tecidos , Animais , Células-Tronco Embrionárias/citologia , Genômica , Gentamicinas/farmacologia , Camundongos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas/genética , Puromicina/farmacologia , Proteínas de Ligação a RNARESUMO
Genomic integrity often is compromised in tumor cells, as illustrated by genetic alterations leading to loss of heterozygosity (LOH). One mechanism of LOH is mitotic crossover recombination between homologous chromosomes, potentially initiated by a double-strand break (DSB). To examine LOH associated with DSB-induced interhomolog recombination, we analyzed recombination events using a reporter in mouse embryonic stem cells derived from F1 hybrid embryos. In this study, we were able to identify LOH events although they occur only rarely in wild-type cells (≤2.5%). The low frequency of LOH during interhomolog recombination suggests that crossing over is rare in wild-type cells. Candidate factors that may suppress crossovers include the RecQ helicase deficient in Bloom syndrome cells (BLM), which is part of a complex that dissolves recombination intermediates. We analyzed interhomolog recombination in BLM-deficient cells and found that, although interhomolog recombination is slightly decreased in the absence of BLM, LOH is increased by fivefold or more, implying significantly increased interhomolog crossing over. These events frequently are associated with a second homologous recombination event, which may be related to the mitotic bivalent structure and/or the cell-cycle stage at which the initiating DSB occurs.
Assuntos
Cromossomos de Mamíferos/genética , Quebras de DNA de Cadeia Dupla , Conversão Gênica/genética , Perda de Heterozigosidade/genética , RecQ Helicases/genética , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Troca Genética/genética , Análise Citogenética , Primers do DNA/genética , Eletroporação , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNARESUMO
The role of bone marrow cells in repairing ectodermal tissue, such as skin epidermis, is not clear. To explore this process further, this study examined a particular form of cutaneous repair, skin grafting. Grafting of full thickness wild-type mouse skin onto mice that had received a green fluorescent protein-bone marrow transplant after whole body irradiation led to an abundance of bone marrow-derived epithelial cells in follicular and interfollicular epidermis that persisted for at least 5 mo. The source of the epithelial progenitors was the nonhematopoietic, platelet-derived growth factor receptor α-positive (Lin(-)/PDGFRα(+)) bone marrow cell population. Skin grafts release high mobility group box 1 (HMGB1) in vitro and in vivo, which can mobilize the Lin(-)/PDGFRα(+) cells from bone marrow to target the engrafted skin. These data provide unique insight into how skin grafts facilitate tissue repair and identify strategies germane to regenerative medicine for skin and, perhaps, other ectodermal defects or diseases.
Assuntos
Células da Medula Óssea/metabolismo , Epiderme/lesões , Epiderme/metabolismo , Proteína HMGB1/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Regeneração , Animais , Transplante de Medula Óssea , Sobrevivência de Enxerto/genética , Proteína HMGB1/genética , Camundongos , Camundongos Transgênicos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Transplante de Pele , Transplante HomólogoRESUMO
The development of haematopoiesis involves the coordinated action of numerous genes, some of which are implicated in haematological malignancies. However, the biological function of many genes remains elusive and unknown functional genes are likely to remain to be uncovered. Here, we report a previously uncharacterised gene in haematopoiesis, identified by screening mutant embryonic stem cells. The gene, 'attenuated haematopoietic development (Ahed)', encodes a nuclear protein. Conditional knockout (cKO) of Ahed results in anaemia from embryonic day 14.5 onward, leading to prenatal demise. Transplantation experiments demonstrate the incapacity of Ahed-deficient haematopoietic cells to reconstitute haematopoiesis in vivo. Employing a tamoxifen-inducible cKO model, we further reveal that Ahed deletion impairs the intrinsic capacity of haematopoietic cells in adult mice. Ahed deletion affects various pathways, and published databases present cancer patients with somatic mutations in Ahed. Collectively, our findings underscore the fundamental roles of Ahed in lifelong haematopoiesis, implicating its association with malignancies.
Assuntos
Hematopoese , Camundongos Knockout , Animais , Hematopoese/genética , Camundongos , Humanos , Feminino , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos Endogâmicos C57BL , Mutação , Anemia/genética , Masculino , Células-Tronco Embrionárias/metabolismoRESUMO
The molecular oscillations underlying the generation of circadian rhythmicity in mammals develop gradually during ontogenesis. However, the developmental process of mammalian cellular circadian oscillator formation remains unknown. In differentiated somatic cells, the transcriptional-translational feedback loops (TTFL) consisting of clock genes elicit the molecular circadian oscillation. Using a bioluminescence imaging system to monitor clock gene expression, we show here that the circadian bioluminescence rhythm is not detected in the mouse embryonic stem (ES) cells, and that the ES cells likely lack TTFL regulation for clock gene expression. The circadian clock oscillation was induced during the differentiation culture of mouse ES cells without maternal factors. In addition, reprogramming of the differentiated cells by expression of Sox2, Klf4, Oct3/4, and c-Myc genes, which were factors to generate induced pluripotent stem (iPS) cells, resulted in the re-disappearance of circadian oscillation. These results demonstrate that an intrinsic program controls the formation of the circadian oscillator during the differentiation process of ES cells in vitro. The cellular differentiation and reprogramming system using cultured ES cells allows us to observe the circadian clock formation process and may help design new strategies to understand the key mechanisms responsible for the organization of the molecular oscillator in mammals.
Assuntos
Diferenciação Celular , Ritmo Circadiano , Células-Tronco Embrionárias/fisiologia , Neurônios/fisiologia , Animais , Relógios Biológicos/genética , Linhagem Celular , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Luminescência , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Tretinoína/farmacologiaRESUMO
Calpains make up a family of Ca(2+)-dependent intracellular cysteine proteases that include ubiquitously expressed µ- and m-calpains. Both are heterodimers consisting of a distinct large catalytic subunit (calpain 1 for µ-calpain and calpain 2 for m-calpain) and a common regulatory subunit (calpain 4). The physiological roles of calpain remain unclear in the organs, including the heart, but it has been suggested that calpain is activated by Ca(2+) overload in diseased hearts, resulting in cardiac dysfunction. In this study, cardiac-specific calpain 4-deficient mice were generated to elucidate the role of calpain in the heart in response to hemodynamic stress. Cardiac-specific deletion of calpain 4 resulted in decreased protein levels of calpains 1 and 2 and showed no cardiac phenotypes under base-line conditions but caused left ventricle dilatation, contractile dysfunction, and heart failure with interstitial fibrosis 1 week after pressure overload. Pressure-overloaded calpain 4-deficient hearts took up a membrane-impermeant dye, Evans blue, indicating plasma membrane disruption. Membrane repair assays using a two-photon laser-scanning microscope revealed that calpain 4-deficient cardiomyocytes failed to reseal a plasma membrane that had been disrupted by laser irradiation. Thus, the data indicate that calpain protects the heart from hemodynamic stresses, such as pressure overload.
Assuntos
Pressão Sanguínea , Calpaína/metabolismo , Insuficiência Cardíaca/enzimologia , Proteínas Musculares/metabolismo , Contração Miocárdica , Miocárdio/enzimologia , Animais , Cálcio/metabolismo , Calpaína/genética , Membrana Celular/enzimologia , Membrana Celular/genética , Membrana Celular/patologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Miocárdio/patologia , Estresse FisiológicoRESUMO
The Na(+)/Ca(2+) exchanger (NCX), an ion-transporter located in the plasma membrane of neuronal cells, contributes to intracellular Ca(2+) homeostasis. Within the brain, three isoforms (NCX1, NCX2, and NCX3) are widely distributed. However, it is not clear to what extent these isoforms are involved in ischemic brain damage in mammals. We therefore used genetically altered mice and isoform-selective NCX inhibitors in a model of transient focal ischemia to investigate the role of each NCX isoform in ischemic brain damage. NCX isoform-mutant mice (NCX1(+/-), NCX2(+/-), and NCX3(+/-)) and wild-type mice were subjected to 90min of middle cerebral artery occlusion (MCAO) followed by 24h of reperfusion. One of three NCX inhibitors [SN-6, KB-R7943, or SEA0400 (3 or 10mgkg(-1), i.p.)] was administered to ddY mice at 30min before more prolonged (4-h) MCAO followed by 24h of reperfusion. After transient MCAO reperfusion, the cerebral infarcts in NCX1(+/-) mice, but not those in NCX2(+/-) or NCX3(+/-) mice, were significantly smaller than those in wild-type mice. SN-6 and SEA0400, which are more selective for the NCX1 isoform, significantly reduced the infarct volume at 10mg/kg. In contrast, KB-R7943, which is more selective for NCX3, did not. These results suggest that the NCX1 isoform may act preferentially (vs. the NCX2 and NCX3 isoforms) to exacerbate the cerebral damage caused by ischemic insult in mice, and that NCX1-selective inhibitors warrant investigation as a potential therapeutic agents for stroke.