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Mammalian meiotic recombination proceeds via repair of hundreds of programmed DNA double-strand breaks, which requires choreographed binding of RPA, DMC1, and RAD51 to single-stranded DNA substrates. High-resolution in vivo binding maps of these proteins provide insights into the underlying molecular mechanisms. When assayed in F1-hybrid mice, these maps can distinguish the broken chromosome from the chromosome used as template for repair, revealing more mechanistic detail and enabling the structure of the recombination intermediates to be inferred. By applying CRISPR-Cas9 mutagenesis directly on F1-hybrid embryos, we have extended this approach to explore the molecular detail of recombination when a key component is knocked out. As a proof of concept, we have generated hybrid biallelic knockouts of Dmc1 and built maps of meiotic binding of RAD51 and RPA in them. DMC1 is essential for meiotic recombination, and comparison of these maps with those from wild-type mice is informative about the structure and timing of critical recombination intermediates. We observe redistribution of RAD51 binding and complete abrogation of D-loop recombination intermediates at a molecular level in Dmc1 mutants. These data provide insight on the configuration of RPA in D-loop intermediates and suggest that stable strand exchange proceeds via multiple rounds of strand invasion with template switching in mouse. Our methodology provides a high-throughput approach for characterization of gene function in meiotic recombination at low animal cost.
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Mouse models with complex genetic backgrounds are increasingly used in preclinical research to accurately model human disease and to enable temporal and cell-specific evaluation of genetic manipulations. Backcrossing mice onto these complex genetic backgrounds takes time and leads to significant wastage of animals. In this study, we aimed to evaluate whether site-specific nucleases could be used to generate additional genetic mutations in a complex genetic background, using the REVERSA mouse model of atherosclerosis, a model harbouring four genetically altered alleles. The model is comprised of a functional null mutation in the Ldlr gene in combination with a ApoB100 allele, which, after high-fat diet, leads to the rapid development of atherosclerosis. The regression of the pathology is achieved by inducible knock-out of the Mttp gene. Here we report an investigation to establish if microinjection of site-specific nucleases directly into zygotes prepared from the REVERSA could be used to investigate the role of the ATP binding cassette transporter G1 (ABCG1) in atherosclerosis regression. We show that using this approach we could successfully generate two independent knockout lines on the REVERSA background, both of which exhibited the expected phenotype of a significant reduction in cholesterol efflux to HDL in bone marrow-derived macrophages. However, loss of Abcg1 did not impact atherosclerosis regression in either the aortic root or in aortic arch, demonstrating no important role for this transporter subtype. We have demonstrated that site-specific nucleases can be used to create genetic modifications directly onto complex disease backgrounds and can be used to explore gene function without the need for laborious backcrossing of independent strains, conveying a significant 3Rs advantage.
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Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis.
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Células Endoteliales/citología , Células Endoteliales/fisiología , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción MEF2/metabolismo , Neovascularización Fisiológica/genética , Animales , Células Cultivadas , Embrión no Mamífero , Células Endoteliales/enzimología , Elementos de Facilitación Genéticos/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factores de Transcripción MEF2/química , Factores de Transcripción MEF2/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Neovascularización Patológica/genética , Dominios y Motivos de Interacción de Proteínas , Retina/embriología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Pez CebraRESUMEN
Sterility or subfertility of male hybrid offspring is commonly observed. This phenomenon contributes to reproductive barriers between the parental populations, an early step in the process of speciation. One frequent cause of such infertility is a failure of proper chromosome pairing during male meiosis. In subspecies of the house mouse, the likelihood of successful chromosome synapsis is improved by the binding of the histone methyltransferase PRDM9 to both chromosome homologs at matching positions. Using genetic manipulation, we altered PRDM9 binding to occur more often at matched sites, and find that chromosome pairing defects can be rescued, not only in an intersubspecific cross, but also between distinct species. Using different engineered variants, we demonstrate a quantitative link between the degree of matched homolog binding, chromosome synapsis, and rescue of fertility in hybrids between Mus musculus and Mus spretus. The resulting partial restoration of fertility reveals additional mechanisms at play that act to lock-in the reproductive isolation between these two species.
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Infertilidad Masculina , Meiosis , Animales , Emparejamiento Cromosómico , Fertilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Infertilidad Masculina/genética , Masculino , Meiosis/genética , RatonesRESUMEN
The DNA-binding protein PRDM9 directs positioning of the double-strand breaks (DSBs) that initiate meiotic recombination in mice and humans. Prdm9 is the only mammalian speciation gene yet identified and is responsible for sterility phenotypes in male hybrids of certain mouse subspecies. To investigate PRDM9 binding and its role in fertility and meiotic recombination, we humanized the DNA-binding domain of PRDM9 in C57BL/6 mice. This change repositions DSB hotspots and completely restores fertility in male hybrids. Here we show that alteration of one Prdm9 allele impacts the behaviour of DSBs controlled by the other allele at chromosome-wide scales. These effects correlate strongly with the degree to which each PRDM9 variant binds both homologues at the DSB sites it controls. Furthermore, higher genome-wide levels of such 'symmetric' PRDM9 binding associate with increasing fertility measures, and comparisons of individual hotspots suggest binding symmetry plays a downstream role in the recombination process. These findings reveal that subspecies-specific degradation of PRDM9 binding sites by meiotic drive, which steadily increases asymmetric PRDM9 binding, has impacts beyond simply changing hotspot positions, and strongly support a direct involvement in hybrid infertility. Because such meiotic drive occurs across mammals, PRDM9 may play a wider, yet transient, role in the early stages of speciation.
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Especiación Genética , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Hibridación Genética/genética , Infertilidad/genética , Ingeniería de Proteínas , Dedos de Zinc/genética , Alelos , Animales , Sitios de Unión , Emparejamiento Cromosómico/genética , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Roturas del ADN de Doble Cadena , Femenino , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Masculino , Meiosis/genética , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Estructura Terciaria de Proteína/genética , Recombinación Genética/genéticaRESUMEN
BACKGROUND: Despite the significant healthcare impact of acute kidney injury, little is known regarding prevention. Single-center data have implicated hypotension in developing postoperative acute kidney injury. The generalizability of this finding and the interaction between hypotension and baseline patient disease burden remain unknown. The authors sought to determine whether the association between intraoperative hypotension and acute kidney injury varies by preoperative risk. METHODS: Major noncardiac surgical procedures performed on adult patients across eight hospitals between 2008 and 2015 were reviewed. Derivation and validation cohorts were used, and cases were stratified into preoperative risk quartiles based upon comorbidities and surgical procedure. After preoperative risk stratification, associations between intraoperative hypotension and acute kidney injury were analyzed. Hypotension was defined as the lowest mean arterial pressure range achieved for more than 10 min; ranges were defined as absolute (mmHg) or relative (percentage of decrease from baseline). RESULTS: Among 138,021 cases reviewed, 12,431 (9.0%) developed postoperative acute kidney injury. Major risk factors included anemia, estimated glomerular filtration rate, surgery type, American Society of Anesthesiologists Physical Status, and expected anesthesia duration. Using such factors and others for risk stratification, patients with low baseline risk demonstrated no associations between intraoperative hypotension and acute kidney injury. Patients with medium risk demonstrated associations between severe-range intraoperative hypotension (mean arterial pressure less than 50 mmHg) and acute kidney injury (adjusted odds ratio, 2.62; 95% CI, 1.65 to 4.16 in validation cohort). In patients with the highest risk, mild hypotension ranges (mean arterial pressure 55 to 59 mmHg) were associated with acute kidney injury (adjusted odds ratio, 1.34; 95% CI, 1.16 to 1.56). Compared with absolute hypotension, relative hypotension demonstrated weak associations with acute kidney injury not replicable in the validation cohort. CONCLUSIONS: Adult patients undergoing noncardiac surgery demonstrate varying associations with distinct levels of hypotension when stratified by preoperative risk factors. Specific levels of absolute hypotension, but not relative hypotension, are an important independent risk factor for acute kidney injury.
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Lesión Renal Aguda/complicaciones , Lesión Renal Aguda/epidemiología , Hipotensión/complicaciones , Hipotensión/epidemiología , Complicaciones Posoperatorias/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anemia/complicaciones , Presión Arterial , Estudios de Cohortes , Femenino , Humanos , Complicaciones Intraoperatorias/epidemiología , Masculino , Persona de Mediana Edad , Periodo Preoperatorio , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Resultado del Tratamiento , Adulto JovenRESUMEN
Mutations in the gene encoding the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS), clinically and pathologically indistinguishable from the majority of 'sporadic' cases of ALS, establishing altered TDP-43 function and distribution as a primary mechanism of neurodegeneration. Transgenic mouse models in which TDP-43 is overexpressed only partially recapitulate the key cellular pathology of human ALS, but may also lead to non-specific toxicity. To avoid the potentially confounding effects of overexpression, and to maintain regulated spatio-temporal and cell-specific expression, we generated mice in which an 80â¯kb genomic fragment containing the intact human TDP-43 locus (either TDP-43WT or TDP-43M337V) and its regulatory regions was integrated into the Rosa26 (Gt(ROSA26)Sor) locus in a single copy. At 3â¯months of age, TDP-43M337V mice are phenotypically normal but by around 6â¯months develop progressive motor function deficits associated with loss of neuromuscular junction integrity, leading to a reduced lifespan. RNA sequencing shows that widespread mis-splicing is absent prior to the development of a motor phenotype, though differential expression analysis reveals a distinct transcriptional profile in pre-symptomatic TDP-43M337V spinal cords. Despite the presence of clear motor abnormalities, there was no evidence of TDP-43 cytoplasmic aggregation in vivo at any timepoint. In primary embryonic spinal motor neurons and in embryonic stem cell (ESC)-derived motor neurons, mutant TDP-43 undergoes cytoplasmic mislocalisation, and is associated with altered stress granule assembly and dynamics. Overall, this mouse model provides evidence that ALS may arise through acquired TDP-43 toxicity associated with defective stress granule function. The normal phenotype until 6â¯months of age can facilitate the study of early pathways underlying ALS.
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Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/genética , Neuronas Motoras/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Fuerza de la Mano , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/patología , Mutación , Unión Neuromuscular/patología , Proteínas de Unión al ARN/metabolismo , Prueba de Desempeño de Rotación con Aceleración ConstanteRESUMEN
The discovery of genetic variants influencing sleep patterns can shed light on the physiological processes underlying sleep. As part of a large clinical sequencing project, WGS500, we sequenced a family in which the two male children had severe developmental delay and a dramatically disturbed sleep-wake cycle, with very long wake and sleep durations, reaching up to 106-h awake and 48-h asleep. The most likely causal variant identified was a novel missense variant in the X-linked GRIA3 gene, which has been implicated in intellectual disability. GRIA3 encodes GluA3, a subunit of AMPA-type ionotropic glutamate receptors (AMPARs). The mutation (A653T) falls within the highly conserved transmembrane domain of the ion channel gate, immediately adjacent to the analogous residue in the Grid2 (glutamate receptor) gene, which is mutated in the mouse neurobehavioral mutant, Lurcher. In vitro, the GRIA3(A653T) mutation stabilizes the channel in a closed conformation, in contrast to Lurcher. We introduced the orthologous mutation into a mouse strain by CRISPR-Cas9 mutagenesis and found that hemizygous mutants displayed significant differences in the structure of their activity and sleep compared to wild-type littermates. Typically, mice are polyphasic, exhibiting multiple sleep bouts of sleep several minutes long within a 24-h period. The Gria3A653T mouse showed significantly fewer brief bouts of activity and sleep than the wild-types. Furthermore, Gria3A653T mice showed enhanced period lengthening under constant light compared to wild-type mice, suggesting an increased sensitivity to light. Our results suggest a role for GluA3 channel activity in the regulation of sleep behavior in both mice and humans.
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Discapacidad Intelectual/genética , Mutación Puntual , Receptores AMPA/genética , Receptores AMPA/metabolismo , Trastornos del Sueño-Vigilia/genética , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Although dantrolene effectively treats malignant hyperthermia (MH), discrepant recommendations exist concerning dantrolene availability. Whereas Malignant Hyperthermia Association of the United States guidelines state dantrolene must be available within 10 min of the decision to treat MH wherever volatile anesthetics or succinylcholine are administered, a Society for Ambulatory Anesthesia protocol permits Class B ambulatory facilities to stock succinylcholine for airway rescue without dantrolene. The authors investigated (1) succinylcholine use rates, including for airway rescue, in anesthetizing/sedating locations; (2) whether succinylcholine without volatile anesthetics triggers MH warranting dantrolene; and (3) the relationship between dantrolene administration and MH morbidity/mortality. METHODS: The authors performed focused analyses of the Multicenter Perioperative Outcomes Group (2005 through 2016), North American MH Registry (2013 through 2016), and Anesthesia Closed Claims Project (1970 through 2014) databases, as well as a systematic literature review (1987 through 2017). The authors used difficult mask ventilation (grades III and IV) as a surrogate for airway rescue. MH experts judged dantrolene treatment. For MH morbidity/mortality analyses, the authors included U.S. and Canadian cases that were fulminant or scored 20 or higher on the clinical grading scale and in which volatile anesthetics or succinylcholine were given. RESULTS: Among 6,368,356 queried outcomes cases, 246,904 (3.9%) received succinylcholine without volatile agents. Succinylcholine was used in 46% (n = 710) of grade IV mask ventilation cases (median dose, 100 mg, 1.2 mg/kg). Succinylcholine without volatile anesthetics triggered 24 MH cases, 13 requiring dantrolene. Among 310 anesthetic-triggered MH cases, morbidity was 20 to 37%. Treatment delay increased complications every 10 min, reaching 100% with a 50-min delay. Overall mortality was 1 to 10%; 15 U.S. patients died, including 4 after anesthetics in freestanding facilities. CONCLUSIONS: Providers use succinylcholine commonly, including during difficult mask ventilation. Succinylcholine administered without volatile anesthetics may trigger MH events requiring dantrolene. Delayed dantrolene treatment increases the likelihood of MH complications. The data reported herein support stocking dantrolene wherever succinylcholine or volatile anesthetics may be used.
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Dantroleno/uso terapéutico , Hipertermia Maligna/tratamiento farmacológico , Hipertermia Maligna/etiología , Relajantes Musculares Centrales/uso terapéutico , Fármacos Neuromusculares Despolarizantes/efectos adversos , Succinilcolina/efectos adversos , Bases de Datos Factuales , HumanosRESUMEN
PURPOSE: Despite growing evidence supporting the potential benefits of higher end-tidal carbon dioxide (ETCO2) levels in surgical patients, there is still insufficient data to formulate guidelines for ideal intraoperative ETCO2 targets. As it is unclear which intraoperative ETCO2 levels are currently used and whether these levels have changed over time, we investigated the practice pattern using the Multicenter Perioperative Outcomes Group database. METHODS: This retrospective, observational, multicentre study included 317,445 adult patients who received general anesthesia for non-cardiothoracic procedures between January 2008 and September 2016. The primary outcome was a time-weighted average area-under-the-curve (TWA-AUC) for four ETCO2 thresholds (< 28, < 35, < 45, and > 45 mmHg). Additionally, a median ETCO2 was studied. A Kruskal-Wallis test was used to analyse differences between years. Random-effect multivariable logistic regression models were constructed to study variability. RESULTS: Both TWA-AUC and median ETCO2 showed a minimal increase in ETCO2 over time, with a median [interquartile range] ETCO2 of 33 [31.0-35.0] mmHg in 2008 and 35 [33.0-38.0] mmHg in 2016 (P <0.001). A large inter-hospital and inter-provider variability in ETCO2 were observed after adjustment for patient characteristics, ventilation parameters, and intraoperative blood pressure (intraclass correlation coefficient 0.36; 95% confidence interval, 0.18 to 0.58). CONCLUSIONS: Between 2008 and 2016, intraoperative ETCO2 values did not change in a clinically important manner. Interestingly, we found a large inter-hospital and inter-provider variability in ETCO2 throughout the study period, possibly indicating a broad range of tolerance for ETCO2, or a lack of evidence to support a specific targeted range. Clinical outcomes were not assessed in this study and they should be the focus of future research.
RéSUMé: OBJECTIF: Malgré une accumulation de données probantes suggérant des avantages de taux plus élevés de dioxyde de carbone en fin d'expiration (ETCO2) chez les patients chirurgicaux, nous ne disposons pas encore d'assez de données pour formuler des lignes directrices sur les cibles peropératoires idéales de l'ETCO2. Comme nous ne savons effectivement pas avec certitude quels taux peropératoires d'ETCO2 sont actuellement utilisés et si ces taux ont changé au fil du temps, nous avons étudié l'évolution de la pratique en utilisant la base de données du MPOG (Multicenter Perioperative Outcomes Group). MéTHODES: Cette étude multicentrique rétrospective observationnelle a inclus 317 445 patients adultes ayant reçu une anesthésie générale pour des procédures non cardiothoraciques entre janvier 2008 et septembre 2016. Le critère d'évaluation principal était une aire sous la courbe moyenne pondérée en fonction du temps (ASC-mT) pour quatre seuils d'ETCO2 (< 28, < 35, < 45 et > 45 mmHg). De plus, une ETCO2 médiane a été étudiée. Un test de Kruskal-Wallis a permis d'analyser les différences entre les années. Des modèles de régression logistique multifactorielle à effet aléatoire ont été construits pour étudier la variabilité. RéSULTATS: L'ASC-mT et l'ETCO2 médiane ont montré une augmentation minime de l'ETCO2 au fil du temps, avec une valeur médiane [plage interquartile] de l'ETCO2 de 33 [31,0 à 35,0] mmHg en 2008 et 35 [33,0 à 38,0] mmHg en 2016 (P < 0,001). Une grande variabilité entre les hôpitaux et prestataires de l'ETCO2 a été observée après ajustement pour les caractéristiques des patients, les paramètres de ventilation et la pression artérielle peropératoire (coefficient de corrélation intracatégorie : 0,36; intervalle de confiance à 95 % : 0,18 à 0,58). CONCLUSIONS: Entre 2008 et 2016, les valeurs peropératoires de l'ETCO2 n'ont pas varié d'une manière importante sur le plan clinique. Il est intéressant de noter que nous avons trouvé une grande variabilité de l'ETCO2 entre hôpitaux et prestataires tout au long de la période d'étude témoignant peut-être d'une vaste plage de tolérance de l'ETCO2 ou d'un manque de données probantes pour soutenir une valeur cible spécifique. L'évolution clinique n'a pas été analysée au cours de cette étude et elle devra être le centre d'intérêt de futures recherches.
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Anestesia General , Dióxido de Carbono/metabolismo , Adulto , Anciano , Capnografía , Bases de Datos Factuales , Femenino , Humanos , Laparoscopía , Masculino , Persona de Mediana Edad , Procedimientos Neuroquirúrgicos , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Estudios Retrospectivos , Procedimientos Quirúrgicos RobotizadosAsunto(s)
Infarto del Miocardio/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Interferencia de ARN , Animales , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Infarto del Miocardio/genética , Infarto del Miocardio/terapia , Tratamiento con ARN de Interferencia/métodosRESUMEN
Iron is essential to the cell. Both iron deficiency and overload impinge negatively on cardiac health. Thus, effective iron homeostasis is important for cardiac function. Ferroportin (FPN), the only known mammalian iron-exporting protein, plays an essential role in iron homeostasis at the systemic level. It increases systemic iron availability by releasing iron from the cells of the duodenum, spleen, and liver, the sites of iron absorption, recycling, and storage respectively. However, FPN is also found in tissues with no known role in systemic iron handling, such as the heart, where its function remains unknown. To explore this function, we generated mice with a cardiomyocyte-specific deletion of Fpn. We show that these animals have severely impaired cardiac function, with a median survival of 22 wk, despite otherwise unaltered systemic iron status. We then compared their phenotype with that of ubiquitous hepcidin knockouts, a recognized model of the iron-loading disease hemochromatosis. The phenotype of the hepcidin knockouts was far milder, with normal survival up to 12 mo, despite far greater iron loading in the hearts. Histological examination demonstrated that, although cardiac iron accumulates within the cardiomyocytes of Fpn knockouts, it accumulates predominantly in other cell types in the hepcidin knockouts. We conclude, first, that cardiomyocyte FPN is essential for intracellular iron homeostasis and, second, that the site of deposition of iron within the heart determines the severity with which it affects cardiac function. Both findings have significant implications for the assessment and treatment of cardiac complications of iron dysregulation.
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Proteínas de Transporte de Catión/fisiología , Corazón/fisiología , Homeostasis , Hierro/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
BACKGROUND: Multiple attempts at tracheal intubation are associated with mortality, and successful rescue requires a structured plan. However, there remains a paucity of data to guide the choice of intubation rescue technique after failed initial direct laryngoscopy. The authors studied a large perioperative database to determine success rates for commonly used intubation rescue techniques. METHODS: Using a retrospective, observational, comparative design, the authors analyzed records from seven academic centers within the Multicenter Perioperative Outcomes Group between 2004 and 2013. The primary outcome was the comparative success rate for five commonly used techniques to achieve successful tracheal intubation after failed direct laryngoscopy: (1) video laryngoscopy, (2) flexible fiberoptic intubation, (3) supraglottic airway as part of an exchange technique, (4) optical stylet, and (5) lighted stylet. RESULTS: A total of 346,861 cases were identified that involved attempted tracheal intubation. A total of 1,009 anesthesia providers managed 1,427 cases of failed direct laryngoscopy followed by subsequent intubation attempts (n = 1,619) that employed one of the five studied intubation rescue techniques. The use of video laryngoscopy resulted in a significantly higher success rate (92%; 95% CI, 90 to 93) than other techniques: supraglottic airway conduit (78%; 95% CI, 68 to 86), flexible bronchoscopic intubation (78%; 95% CI, 71 to 83), lighted stylet (77%; 95% CI, 69 to 83), and optical stylet (67%; 95% CI, 35 to 88). Providers most frequently choose video laryngoscopy (predominantly GlideScope [Verathon, USA]) to rescue failed direct laryngoscopy (1,122/1,619; 69%), and its use has increased during the study period. CONCLUSIONS: Video laryngoscopy is associated with a high rescue intubation success rate and is more commonly used than other rescue techniques.
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Intubación Intratraqueal , Laringoscopía , Evaluación de Resultado en la Atención de Salud/estadística & datos numéricos , Atención Perioperativa/métodos , Diseño de Equipo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Insuficiencia del TratamientoRESUMEN
For the analysis of gene function in vivo, gene overexpression in the mouse provides an alternative to loss-of-function knock-out approaches and can help reveal phenotypes where compensatory mechanisms are at play. Furthermore, when multiple lines overexpressing a gene-of-interest at varying levels are studied, the consequences of differences in gene dosage can be explored. Despite these advantages, inherent shortcomings in the methodologies used for the generation of gain-of-function transgenic mouse models have limited their application to functional gene analysis, and the necessity for multiple lines comes at a significant animal and financial cost. The targeting of transgenic overexpression constructs at single copy into neutral genomic loci is the preferred method for the generation of such models, which avoids the unpredictable outcomes associated with conventional random integration. However, despite the increased reliability that targeted transgenic methodologies provide, only one expression level results, as defined by the promoter used. Here, we report a new versatile overexpression allele, the promoter-switch allele, which couples PhiC31 integrase-targeted transgenesis with Flp recombinase promoter switching and Cre recombinase activation. These recombination switches allow the conversion of different overexpression alleles, combining the advantages of transgenic targeting with tunable transgene expression. With this approach, phenotype severity can be correlated with transgene expression in a single mouse model, providing a cost-effective solution amenable to systematic gain-of-function studies.
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Expresión Génica , Transgenes , Alelos , Animales , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Regiones Promotoras GenéticasRESUMEN
Integration of a point mutation to correct or edit a gene requires the repair of the CRISPR-Cas9-induced double-strand break by homology-directed repair (HDR). This repair pathway is more active in late S and G2 phases of the cell cycle, whereas the competing pathway of nonhomologous end-joining (NHEJ) operates throughout the cell cycle. Accordingly, modulation of the cell cycle by chemical perturbation or simply by the timing of gene editing to shift the editing toward the S/G2 phase has been shown to increase HDR rates. Using a traffic light reporter in mouse embryonic stem cells and a fluorescence conversion reporter in human-induced pluripotent stem cells, we confirm that a transient cold shock leads to an increase in the rate of HDR, with a corresponding decrease in the rate of NHEJ repair. We then investigated whether a similar cold shock could lead to an increase in the rate of HDR in the mouse embryo. By analyzing the efficiency of gene editing using single nucleotide polymorphism changes and loxP insertion at three different genetic loci, we found that a transient reduction in temperature after zygote electroporation of CRISPR-Cas9 ribonucleoprotein with a single-stranded oligodeoxynucleotide repair template did indeed increase knockin efficiency, without affecting embryonic development. The efficiency of gene editing with and without the cold shock was first assessed by genotyping blastocysts. As a proof of concept, we then confirmed that the modified embryo culture conditions were compatible with live births by targeting the coat color gene tyrosinase and observing the repair of the albino mutation. Taken together, our data suggest that a transient cold shock could offer a simple and robust way to improve knockin outcomes in both stem cells and zygotes.
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Edición Génica , Hipotermia , Animales , Humanos , Ratones , Sistemas CRISPR-Cas/genética , Cigoto/metabolismo , Hipotermia/metabolismo , Reparación del ADN por Recombinación/genéticaRESUMEN
For the preparation of embryo transfer recipients, surgically vasectomized mice are commonly used, generated by procedures associated with pain and discomfort. Sterile transgenic strains provide a nonsurgical replacement, but their maintenance requires breeding and genotyping procedures. We have previously reported the use of naturally sterile STUSB6F1 hybrids for the production of embryo transfer recipients and found the behavior of these recipients to be indistinguishable from those generated by vasectomized males. The method provides two substantial 3R impacts: refinement (when compared with surgical vasectomy) and reduction in breeding procedures (compared with sterile transgenic lines). Despite initial promise, the 3Rs impact of this innovation was limited by difficulties in breeding the parental STUS/Fore strain, which precluded the wider distribution of the sterile hybrid. The value of a 3R initiative is only as good as the uptake in the community. Here we, thus, select a different naturally sterile hybrid, generated from strains that are widely available: the B6SPRTF1 hybrid between C57BL/6J and Mus spretus. We first confirmed its sterility by sperm counting and testes weight and then trialed the recovery of cryopreserved embryos and germplasm within three UK facilities. Distribution of sperm for the generation of these hybrids by in vitro fertilization was found to be the most robust distribution method and avoided the need to maintain a live M. spretus colony. We then tested the suitability of B6SPRTF1 sterile hybrids for the generation of embryo transfer recipients at these same three UK facilities and found the hybrids to be suitable when compared with surgical vasectomized mice and a sterile transgenic strain. In conclusion, the potential 3Rs impact of this method was confirmed by the ease of distribution and the utility of sterile B6SPRTF1 hybrids at independent production facilities.
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Transferencia de Embrión , Ratones Endogámicos C57BL , Animales , Masculino , Ratones , Transferencia de Embrión/veterinaria , Transferencia de Embrión/métodos , Femenino , Hibridación Genética , Seudoembarazo/genética , Seudoembarazo/veterinaria , Criopreservación/veterinaria , Fertilización In Vitro/veterinaria , Fertilización In Vitro/métodos , Vasectomía/veterinaria , Vasectomía/métodosRESUMEN
Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-ß1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-ß1 followed by COL6A1. Knockdown of TGF-ß1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-ß1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Células Madre Pluripotentes Inducidas , Animales , Humanos , Ratones , Demencia Frontotemporal/patología , Esclerosis Amiotrófica Lateral/metabolismo , Factor de Crecimiento Transformador beta1 , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/metabolismo , Drosophila , Matriz Extracelular/metabolismo , Dipéptidos/metabolismo , Expansión de las Repeticiones de ADN/genéticaRESUMEN
The targeting of transgenic constructs at single copy into neutral genomic loci avoids the unpredictable outcomes associated with conventional random integration approaches. The Gt(ROSA)26Sor locus on chromosome 6 has been used many times for the integration of transgenic constructs and is known to be permissive for transgene expression and disruption of the gene is not associated with a known phenotype. Furthermore, the transcript made from the Gt(ROSA)26Sor locus is ubiquitously expressed and subsequently the locus can be used to drive the ubiquitous expression of transgenes.Here we report a protocol for the generation of targeted transgenic alleles at Gt(ROSA)26Sor, taking as an example a conditional overexpression allele, by PhiC31 integrase/recombinase-mediated cassette exchange of an engineered Gt(ROSA)26Sor locus in mouse embryonic stem cells. The overexpression allele is initially silenced by the presence of a loxP flanked stop sequence but can be strongly activated through the action of Cre recombinase.
Asunto(s)
Rosa , Ratones , Animales , Ratones Transgénicos , Rosa/genética , Transgenes , Integrasas/genética , Integrasas/metabolismo , Recombinasas/genéticaRESUMEN
53BP1 regulates DNA end-joining in lymphocytes, diversifying immune antigen receptors. This involves nucleosome-bound 53BP1 at DNA double-stranded breaks (DSBs) recruiting RIF1 and shieldin, a poorly understood DNA-binding complex. The 53BP1-RIF1-shieldin axis is pathological in BRCA1-mutated cancers, blocking homologous recombination (HR) and driving illegitimate non-homologous end-joining (NHEJ). However, how this axis regulates DNA end-joining and HR suppression remains unresolved. We investigated shieldin and its interplay with CST, a complex recently implicated in 53BP1-dependent activities. Immunophenotypically, mice lacking shieldin or CST are equivalent, with class-switch recombination co-reliant on both complexes. ATM-dependent DNA damage signalling underpins this cooperation, inducing physical interactions between these complexes that reveal shieldin as a DSB-responsive CST adaptor. Furthermore, DNA polymerase ζ functions downstream of shieldin, establishing DNA fill-in synthesis as the physiological function of shieldin-CST. Lastly, 53BP1 suppresses HR and promotes NHEJ in BRCA1-deficient mice and cells independently of shieldin. These findings showcase the resilience of the 53BP1 pathway, achieved through the collaboration of chromatin-bound 53BP1 complexes and DNA end-processing effector proteins.