RESUMEN
In vitro studies have identified LIMK2 as a key downstream effector of Rho GTPase-induced changes in cytoskeletal organization. LIMK2 is phosphorylated and activated by Rho associated coiled-coil kinases (ROCKs) in response to a variety of growth factors. The biochemical targets of LIMK2 belong to a family of actin binding proteins that are potent modulators of actin assembly and disassembly. Although numerous studies have suggested that LIMK2 regulates cell morphology and motility, evidence supportive of these functions in vivo has remained elusive. In this study, a knockout mouse was created that abolished LIMK2 biochemical activity resulting in a profound inhibition of epithelial sheet migration during eyelid development. In the absence of LIMK2, nascent eyelid keratinocytes differentiate and acquire a pre-migratory phenotype but the leading cells fail to nucleate filamentous actin and remain immobile causing an eyes open at birth (EOB) phenotype. The failed nucleation of actin was associated with significant reductions in phosphorylated cofilin, a major LIMK2 biochemical substrate and potent modulator of actin dynamics. These results demonstrate that LIMK2 activity is required for keratinocyte migration in the developing eyelid.
Asunto(s)
Movimiento Celular/genética , Párpados/citología , Queratinocitos/citología , Quinasas Lim/fisiología , Actinas/metabolismo , Animales , Párpados/embriología , Párpados/patología , Genotipo , Quinasas Lim/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , FosforilaciónRESUMEN
Large collections of knockout organisms facilitate the elucidation of gene functions. Here we used retroviral insertion or homologous recombination to disrupt 472 genes encoding secreted and membrane proteins in mice, providing a resource for studying a large fraction of this important class of drug target. The knockout mice were subjected to a systematic phenotypic screen designed to uncover alterations in embryonic development, metabolism, the immune system, the nervous system and the cardiovascular system. The majority of knockout lines exhibited altered phenotypes in at least one of these therapeutic areas. To our knowledge, a comprehensive phenotypic assessment of a large number of mouse mutants generated by a gene-specific approach has not been described previously.
Asunto(s)
Proteínas de la Membrana/genética , Animales , Ratones , Ratones NoqueadosRESUMEN
PURPOSE: Goals of this study were to determine if pharmacological or genetic inhibition of Rho-associated coiled coil containing protein kinases (known as ROCK1 and ROCK2) alters intraocular pressure (IOP) in mice. METHODS: Micro-cannulation of the anterior chamber was used to measure IOP in wild-type B6.129 hybrid mice following treatment with ROCK inhibitors Y-27632 or Y-39983. For comparative purposes, wild-type mice were also treated with timolol, acetazolamide, pilocarpine, or latanoprost. Mice deficient in either Rock1 or Rock2 were generated by homologous recombination or gene trapping, respectively, and their IOP was determined using identical methods employed in the pharmacology studies. RESULTS: Treatment of wild-type B6.129 hybrid mice with ROCK inhibitors (Y-27632 and Y-39983) resulted in significant reductions in IOP. The magnitude of IOP reduction observed with topical Y-39983 was comparable to timolol, and exceeded the IOP effects of latanoprost in this study. Pilocarpine had no discernible effect on IOP in mice. Moreover, mice deficient in either Rock1 or Rock2 exhibited a significant decrease in IOP compared to their B6.129 wild-type littermates. CONCLUSIONS: Pharmacological or genetic inhibition of ROCKs results in decreased IOP in mice. The magnitude of IOP reduction is significant as demonstrated with comparative pharmacology using agents that lower IOP in humans. These studies support the ROCK pathway as a therapeutic target for treating ocular hypertension.
Asunto(s)
Amidas/farmacología , Presión Intraocular/efectos de los fármacos , Piridinas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genética , Acetazolamida/administración & dosificación , Acetazolamida/farmacología , Administración Tópica , Antagonistas Adrenérgicos beta/administración & dosificación , Antagonistas Adrenérgicos beta/farmacología , Amidas/administración & dosificación , Animales , Antihipertensivos/administración & dosificación , Antihipertensivos/farmacología , Relación Dosis-Respuesta a Droga , Latanoprost , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Agonistas Muscarínicos/administración & dosificación , Agonistas Muscarínicos/farmacología , Pilocarpina/administración & dosificación , Pilocarpina/farmacología , Prostaglandinas F Sintéticas/administración & dosificación , Prostaglandinas F Sintéticas/farmacología , Piridinas/administración & dosificación , Timolol/administración & dosificación , Timolol/farmacologíaRESUMEN
P2Y receptors have been reported to modulate gastrointestinal functions. The newest family member is the nucleotide-sugar receptor P2Y14. P2ry14 mRNA was detected throughout the rat gut, with the highest level being in the forestomach. We investigated the role of the receptor in stomach motility using cognate agonists and knockout (KO) mice. In rat isolated forestomach, 100 microM UDP-glucose and 100 muM UDP-galactose both increased the baseline muscle tension (BMT) by 6.2+/-0.6 and 1.6+/-0.6 mN (P<0.05, n=3-4), respectively, and the amplitude of contractions during electrical field stimulation (EFS) by 3.7+/-1.7 and 4.3+/-2.5 mN (P<0.05, n=3-4), respectively. In forestomach from wild-type (WT) mice, 100 microM UDP-glucose increased the BMT by 1.0+/-0.1 mN (P<0.05, n=6) but this effect was lost in the KO mice (change of -0.1+/-0.1 mN, n=6). The 100 microM UDP-glucose also increased the contraction amplitude during EFS in this tissue from the WT animals (0.9+/-0.4 mN, P < 0.05, n=6) but not from the KO mice (0.0+/-0.2 mN, n=6). In vivo, UDP-glucose at 2,000 mg/kg ip reduced gastric emptying in rats by 49.7% (P<0.05, n=4-6) and in WT and KO mice by 56.1 and 66.2%, respectively (P<0.05, n=7-10) vs. saline-treated control animals. There was no significant difference in gastric emptying between WT and KO animals receiving either saline or d-glucose. These results demonstrate a novel function of the P2Y14 receptor associated with contractility in the rodent stomach that does not lead to altered gastric emptying after receptor deletion and an ability of UDP-glucose to delay gastric emptying without involving the P2Y14 receptor.
Asunto(s)
Vaciamiento Gástrico/efectos de los fármacos , Receptores Purinérgicos P2/metabolismo , Uridina Difosfato Glucosa/farmacología , Animales , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/fisiología , Operón Lac/genética , Operón Lac/fisiología , Ratones , Ratones Noqueados , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y , Uridina Difosfato Galactosa/farmacologíaRESUMEN
We report the construction and analysis of a mouse gene trap mutant resource created in the C57BL/6N genetic background containing more than 350,000 sequence-tagged embryonic stem (ES) cell clones. We also demonstrate the ability of these ES cell clones to contribute to the germline and produce knockout mice. Each mutant clone is identified by a genomic sequence tag representing the exact insertion location, allowing accurate prediction of mutagenicity and enabling direct genotyping of mutant alleles. Mutations have been identified in more than 10,000 genes and show a bias toward the first intron. The trapped ES cell lines, which can be requested from the Texas A&M Institute for Genomic Medicine, are readily available to the scientific community.
Asunto(s)
Células Madre Embrionarias/metabolismo , Mutagénesis Insercional , Animales , Blastocisto/metabolismo , Línea Celular , Quimera , Células Clonales , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Intrones , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , MicroARNsRESUMEN
ADP-ribosylation factor-like 3 (Arl3) is a member of a small subfamily of G-proteins involved in membrane-associated vesicular and intracellular trafficking processes. Genetic studies in Leishmania have shown that the Arl3 homolog is essential for flagellum biogenesis. Mutations in a related human family member, Arl6, result in Bardet-Biedl syndrome in humans, which is characterized by genital, renal, and retinal abnormalities, obesity, and learning deficits. As part of our large-scale phenotypic screen, mice deficient for the Arl3 gene were generated and analyzed. Arl3 (-/-) mice were born at a sub-Mendelian ratio, were small and sickly, and had markedly swollen abdomens. These mutants failed to thrive, and all died by 3 weeks of age. The (-/-) mice exhibited abnormal development of renal, hepatic, and pancreatic epithelial tubule structures, which is characteristic of the renal-hepatic-pancreatic dysplasia found in autosomal recessive polycystic kidney disease. Absence of Arl3 was associated with abnormal epithelial cell proliferation and cyst formation. Moreover, mice lacking Arl3 exhibited photoreceptor degeneration as early as postnatal day 14. These results are the first to implicate Arl3 in a ciliary disease affecting the kidney, biliary tract, pancreas, and retina.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Apoptosis , Riñón/crecimiento & desarrollo , Células Fotorreceptoras de Vertebrados/fisiología , Factores de Ribosilacion-ADP/genética , Animales , Sistema Biliar/anomalías , Femenino , Antígeno Ki-67/metabolismo , Riñón/metabolismo , Masculino , Ratones , Ratones Noqueados , Mutación , Páncreas/crecimiento & desarrollo , Páncreas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/crecimiento & desarrollo , Retina/metabolismoRESUMEN
The serine protease HtrA2/Omi is released from the mitochondrial intermembrane space following apoptotic stimuli. Once in the cytosol, HtrA2/Omi has been implicated in promoting cell death by binding to inhibitor of apoptosis proteins (IAPs) via its amino-terminal Reaper-related motif, thus inducing caspase activity, and also in mediating caspase-independent death through its own protease activity. We report here the phenotype of mice entirely lacking expression of HtrA2/Omi due to targeted deletion of its gene, Prss25. These animals, or cells derived from them, show no evidence of reduced rates of cell death but on the contrary suffer loss of a population of neurons in the striatum, resulting in a neurodegenerative disorder with a parkinsonian phenotype that leads to death of the mice around 30 days after birth. The phenotype of these mice suggests that it is the protease function of this protein and not its IAP binding motif that is critical. This conclusion is reinforced by the finding that simultaneous deletion of the other major IAP binding protein, Smac/DIABLO, does not obviously alter the phenotype of HtrA2/Omi knockout mice or cells derived from them. Mammalian HtrA2/Omi is therefore likely to function in vivo in a manner similar to that of its bacterial homologues DegS and DegP, which are involved in protection against cell stress, and not like the proapoptotic Reaper family proteins in Drosophila melanogaster.
Asunto(s)
Cuerpo Estriado/embriología , Cuerpo Estriado/enzimología , Serina Endopeptidasas/fisiología , Animales , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Cuerpo Estriado/anomalías , ADN/genética , Femenino , Marcación de Gen , Serina Peptidasa A2 que Requiere Temperaturas Altas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/fisiología , Neuronas/patología , Trastornos Parkinsonianos/embriología , Trastornos Parkinsonianos/etiología , Trastornos Parkinsonianos/genética , Fenotipo , Embarazo , Proteínas/metabolismo , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
Glycogen synthase kinase-3 (GSK-3) protein levels and activity are elevated in skeletal muscle in type 2 diabetes, and inversely correlated with both glycogen synthase activity and insulin-stimulated glucose disposal. To explore this relationship, we have produced transgenic mice that overexpress human GSK-3beta in skeletal muscle. GSK-3beta transgenic mice were heavier, by up to 20% (P < .001), than their age-matched controls due to an increase in fat mass. The male GSK-3beta transgenic mice had significantly raised plasma insulin levels and by 24 weeks of age became glucose-intolerant as determined by a 50% increase in the area under their oral glucose tolerance curve (P < .001). They were also hyperlipidemic with significantly raised serum cholesterol (+90%), nonesterified fatty acids (NEFAs) (+55%), and triglycerides (+170%). At 29 weeks of age, GSK-3beta protein levels were 5-fold higher, and glycogen synthase activation (-27%), glycogen levels (-58%) and insulin receptor substrate-1 (IRS-1) protein levels (-67%) were significantly reduced in skeletal muscle. Hepatic glycogen levels were significantly increased 4-fold. Female GSK-3beta transgenic mice did not develop glucose intolerance despite 7-fold overexpression of GSK-3beta protein and a 20% reduction in glycogen synthase activation in skeletal muscle. However, plasma NEFAs and muscle IRS-1 protein levels were unchanged in females. We conclude that overexpression of human GSK-3beta in skeletal muscle of male mice resulted in impaired glucose tolerance despite raised insulin levels, consistent with the possibility that elevated levels of GSK-3 in type 2 diabetes are partly responsible for insulin resistance.
Asunto(s)
Intolerancia a la Glucosa/genética , Glucógeno Sintasa Quinasa 3/biosíntesis , Glucógeno Sintasa Quinasa 3/genética , Músculo Esquelético/fisiología , Regiones Promotoras Genéticas/fisiología , Animales , Western Blotting , Composición Corporal/fisiología , Peso Corporal/fisiología , Cartilla de ADN , ADN Complementario/biosíntesis , ADN Complementario/genética , Femenino , Prueba de Tolerancia a la Glucosa , Glucógeno/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Proteínas Sustrato del Receptor de Insulina , Resistencia a la Insulina/genética , Resistencia a la Insulina/fisiología , Lípidos/sangre , Hígado/metabolismo , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , Fenotipo , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.
Asunto(s)
Presión Sanguínea/fisiología , Proteínas Serina-Treonina Quinasas/deficiencia , Animales , Secuencia de Bases , Presión Sanguínea/genética , ADN Complementario/genética , Biblioteca de Genes , Técnicas Genéticas , Heterocigoto , Humanos , Hipertensión/terapia , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Mutagénesis Insercional/métodos , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Lugares Marcados de Secuencia , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
The ability of native uncoupling protein-3 (UCP3) to uncouple mitochondrial oxidative phosphorylation is controversial. We measured the expression level of UCP3 and the proton conductance of skeletal muscle mitochondria isolated from transgenic mice overexpressing human UCP3 (UCP3-tg) and from UCP3 knockout (UCP3-KO) mice. The concentration of UCP3 in UCP3-tg mitochondria was approximately 3 microg/mg protein, approximately 20-fold higher than the wild type value. UCP3-tg mitochondria had increased nonphosphorylating respiration rates, decreased respiratory control, and approximately 4-fold increased proton conductance compared with the wild type. However, this increased uncoupling in UCP3-tg mitochondria was not caused by native function of UCP3 because it was not proportional to the increase in UCP3 concentration and was neither activated by superoxide nor inhibited by GDP. UCP3 was undetectable in mitochondria from UCP3-KO mice. Nevertheless, UCP3-KO mitochondria had unchanged respiration rates, respiratory control ratios, and proton conductance compared with the wild type under a variety of assay conditions. We conclude that uncoupling in UCP3-tg mice is an artifact of transgenic expression, and that UCP3 does not catalyze the basal proton conductance of skeletal muscle mitochondria in the absence of activators such as superoxide.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Protones , Animales , Western Blotting , Peso Corporal , Humanos , Canales Iónicos , Cinética , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Mitocondriales , Oxígeno/metabolismo , Consumo de Oxígeno , Fosforilación , Unión Proteica , Ácido Succínico/metabolismo , Superóxidos/metabolismo , Proteína Desacopladora 3RESUMEN
The completion of the Human Genome Project has signaled the beginning of the post-genome era, with a corresponding shift in focus from the sequencing and identification of genes to the exploration of gene function. A rate-limiting step in deriving value from this gene sequence information is determining the potential pharmaceutical applications of genes and their encoded proteins. This validation step is crucial for focusing efforts and resources on only the most promising targets. Strategies using reverse mouse genetics provide excellent methods for validating potential targets and therapeutic proteins in vivo in a mammalian model system.