Los autores agradecen al personal del núcleo Morfología e Imagen, del núcleo de Genética Molecular (Departamento de Oftalmología y Ciencias Visuales) y del ratón Genética Core en la Universidad de Washington en St. Louis Escuela de Medicina. Los autores son apoyados por el programa de pequeñas donaciones del centro McDonnell Celular y Neurobiología Molecular, el Centro Esperanza de Trastornos Neurológicos, el Instituto Nacional del Ojo (# R01EY022632 a DH), un Instituto Oftalmológico Centro Nacional Core Grant (# P30EY002687) y un subvención sin restricciones de Investigación de Prevención de la Ceguera en el Departamento de Oftalmología y Ciencias Visuales.

Declaración de Ética: Procedimientos en seres animales fueron aprobadas por el Cuidado y Uso de Animales Comité Institucional (IACUC) de la Universidad de Washington en St. Louis. 1. Ratón Cría y Genotipado <ol><li> Raza ratones Tg Tg (CAG-LacZ / EGFP-KASH2) (PCP2-Cre) ratones para producir Tg (PCP2Cre CAG-EGFP / KASH2) 19,20. Nota: Mientras que el resto de este protocolo se centra en el uso de la línea de Tg (PCP2-Cre) del ratón para restringir EGFP-KASH2 expresión a las células de Purkinje en el cerebelo, un procedimiento similar puede ser seguido con cualquier otro Tg (Cre) ratón ( Figura 2A). </li><li> Realizar genotipado para identificar los cachorros que albergan ambos transgenes EGFP y Cre como se describió anteriormente 19. </li></ol> 2. Preparación de los materiales para la disección y Tissue Collection <ol><li> Preparar 100 ml de 30% de sacarosa en PBS (30 g de sacarosa / 100 ml de PBS 1x) y se almacena a -20 ° C. El día de tejido harvesting, deshielo y la carga ~ 30-35 ml de solución de sacarosa en una jeringa de 35 ml para perfusiones transcardial. </li><li> Preparar 40 ml de PFA al 4% en PBS (10 ml 16% de paraformaldehído y 30 ml de PBS). Cargar ~ de 30-35 ml de PFA al 4% en una jeringa de 35 ml para perfusiones transcardial. </li><li> Llenar una placa de cultivo tisular de 10 cm con 10 ml de PBS para la disección de tejidos. </li><li> Preparar un cóctel de ketamina / xilazina que consiste en 16 mg / ml de ketamina y 3 mg / ml de xilazina. </li><li> Preparar una solución de etanol al 70% para la disección. </li></ol> 3. La disección y Tissue Collection <ol><li> Usando una jeringa de 1 ml, inyectar 500 l de cóctel de ketamina el / xilazina por cada 25 gramos de peso corporal y esperar a ~ 15 min. Alternativamente, utilizar otro método aprobado institucionalmente sedación que es apropiado para el sistema biológico de interés. </li><li> Tras la sedación, pellizque suavemente la pata trasera para confirmar la falta de respuesta al dolor, fijar todas las patas en una bandeja de espuma de poliestireno con la cara dorsal de ladel ratón hacia abajo. </li><li> Pulverizar el abdomen con 70% de etanol y hacer una incisión desde el abdomen inferior a la parte superior de la caja torácica para obtener acceso al corazón. </li><li> Hacer una pequeña incisión en la aurícula derecha del corazón y de insertar la jeringa que contiene la solución de sacarosa al 30% en el ventrículo izquierdo del corazón. </li><li> Perfundir la solución a una velocidad de ~ 2 a 5 ml por min. Cuando está vacío, retire la jeringa y repita con la solución de PFA 4%. </li><li> Liberar el ratón de la bandeja de espuma de poliestireno y decapitar para acceder al cerebro. </li><li> Retire la piel alrededor de la cabeza del animal con unas pinzas y tijeras. Haga un agujero en la parte posterior del cráneo con una aguja y retire con cuidado el cráneo con tijeras y pinzas de disección. </li><li> Una vez que el cerebro es claramente accesible, utilizar pinzas para quitar el cerebelo y colocarlo en PBS. Dividir en dos el cerebelo en el plano sagital con un escalpelo y transferir a la solución de sacarosa al 30% durante la noche a 4 ° C. Nota: Para prepararson secciones de parafina, fijar el cerebelo durante la noche a 4 ° C en el 4% PFA antes de la incrustación de tejidos y seccionamiento. </li></ol> 4. Procesamiento de tejidos y Seccionamiento <ol><li> Preparar una suspensión de hielo seco y butano 2-metilo. Dejar que la temperatura del baño se equilibre durante 5 min. </li><li> Llene una criomolde con compuesto de octubre y coloque una media del cerebelo biseccionado en el molde. </li><li> Usando un microscopio de disección y un par de fórceps posición el cerebelo manera que la superficie dividida en dos partes (cerca de la línea media del cerebelo) quede hacia arriba. </li><li> Transferir el criomolde a la suspensión de hielo seco durante 5 min hasta que el compuesto OCT ha formado un sólido blanco alrededor de matriz de la muestra de tejido. Nota: la muestra congelada ahora se pueden almacenar a -20 ° C si es necesario. </li><li> Label diapositivas poli-lisina pre-tratada para la recogida de las secciones de tejido y preparar el criostato de acuerdo con las instrucciones del fabricante con el fin de recoger 15 micras secciones de la cerebellum. Para recoger las secciones, póngase en contacto con atención la sección de octubre con el lado revestido de poli-lisina de la diapositiva. Para preservar la orientación de la sección, no gire la diapositiva durante este paso. </li><li> Secciones de tejido tienda a -20 ° C hasta proceder a la tinción y de imagen. </li></ol> 5. La tinción e Imagen de Cerebelo Secciones <ol><li> Preparar las siguientes soluciones antes de la tinción de tejidos: ~ 50 ml de PBS ~ 5-10 ml de PFA al 4% en PBS ~ 10-20 ml de 0,5% Triton X-100, 10% de suero de burro en PBS </li><li> Secciones de cerebelo deshielo. Es importante destacar que minimizar su exposición a cualquier fuente de luz continua e intensa para prevenir la decoloración de EGFP. </li><li> El uso de una barrera hidrofóbica lápiz, dibujar un rectángulo alrededor de las secciones de tejido. </li><li> Post-fijar las secciones con 4% PFA durante 5 min. Use suficiente líquido para cubrir toda el área delimitada por la barrera hidrofóbica (normalmente ~ 500 l). El uso de un vacío o pipeta, remover el fijador particularmente cuidado de no molestar a los cortes de tejido. </li><li> Con una pipeta, aplicar suavemente PBS sobre el portaobjetos sin molestar a las secciones de tejido. Después de 5 min utilizar el vacío o pipeta para retirar con cuidado el líquido. Repita el enjuague de PBS un total de tres veces. </li><li> Con una pipeta, aplicar cuidadosamente la dilución adecuada de anticuerpos primarios (3 l Calbindin y / o anticuerpo Nesprin2 por 900 l de solución) en 0,5% de Triton X-100, 10% de suero de burro en PBS. Incubar durante una hora a temperatura ambiente en la oscuridad. Nota: EGFP-KASH2 será visible por microscopía de fluorescencia directa sobre secciones congeladas, y no es necesario utilizar un anticuerpo anti-EGFP. Sin embargo, si se han preparado las secciones de parafina, formación de imágenes de EGFP-KASH2 requiere inmunodetección con un anticuerpo anti-EGFP. </li><li> Retire la solución de anticuerpo y enjuague con PBS tres veces. </li><li> Preparar 1: 1.000 diluciones de Alexa-Fluor anticuerpos secundarios en 0,5% Triton X-100,10% de suero de burro en PBS y almacenar en la oscuridad. </li><li> Retire con cuidado el enjuague final PBS y aplicar la dilución de anticuerpo secundario durante una hora en la oscuridad. </li><li> Retire la solución de anticuerpo secundario, aclarar y lavar la sección tres veces. Si es necesario, aplicar una mancha nuclear, tales como DAPI (~ 300 nM), durante la primera aplicación PBS. </li><li> Completamente eliminar la solución PBS de todo el tejido y el lugar ~ 5.7 l de medios de montaje en la sección de tejido. Coloque un cubreobjetos de vidrio en la parte superior de la sección de tejido y dejar de lado por unos 15-30 minutos a temperatura ambiente en la oscuridad. </li><li> Utilice un microscopio de fluorescencia equipado con los filtros apropiados para visualizar EGFP y cualesquiera otras longitudes de onda adecuadas para los anticuerpos secundarios utilizados en los pasos 5.8 / 5.9. En rebanadas del cerebelo, utilice un objetivo 20X para ver llantas nucleares EGFP-KASH2. </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nesprins+anchor+kinesin-1+motors+to+the+nucleus+to+drive+nuclear+distribution+in+muscle+cells.">Nesprins anchor kinesin-1 motors to the nucleus to drive nuclear distribution in muscle cells.</a> Development. 142, 218-228 (2015).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KASH+protein+Syne-2/Nesprin-2+and+SUN+proteins+SUN1/2+mediate+nuclear+migration+during+mammalian+retinal+development.">KASH protein Syne-2/Nesprin-2 and SUN proteins SUN1/2 mediate nuclear migration during mammalian retinal development.</a> Human molecular genetics. 20, 1061-1073 (2011).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syne-1+and+Syne-2+play+crucial+roles+in+myonuclear+anchorage+and+motor+neuron+innervation.">Syne-1 and Syne-2 play crucial roles in myonuclear anchorage and motor neuron innervation.</a> Development. 134, 901-908 (2007).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nesprin+1+is+critical+for+nuclear+positioning+and+anchorage.">Nesprin 1 is critical for nuclear positioning and anchorage.</a> Human molecular genetics. 19, 329-341 (2010).</li><li>Stewart-Hutchinson, P. 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Los autores no tienen nada que revelar.

El paso más crítico para estudiar con éxito el papel de los complejos de LINC in vivo utilizando el modelo de Tg (CAG-LacZ / EGFP-KASH2) es identificar una línea de ratones Cre adecuado (s). En efecto, si Cre está activo en otros tipos de células implicados en las vías similares, se puede complicar la interpretación de los resultados. Por lo tanto, es importante examinar las células cercanas, como se muestra aquí en las capas celulares moleculares y granulares del cerebelo (Figura 2B).</strong...

La envoltura nuclear (NE) separa el nucleoplasma del citoplasma. Se compone de una membrana nuclear interna y externa (INM y ONM, respectivamente) que se conectan en los poros nucleares. El lumen delimitado por ambas membranas se llama el espacio perinuclear (PNS). La ONM es una extensión del retículo endoplásmico rugoso (ER), y el INM se adhiere a la lámina nuclear, una malla de tipo nuclear-V filamentos intermedios representado por A y de tipo B laminas 1,2. Enlazadores de los complejos Citoesqueleto (LINC) nucleoesqueleto y son conjuntos macromoleculares que abarcan toda la envoltura nuclear para conectar físicamente el interior del núcleo a los filamentos del citoesqueleto y motores moleculares (Figura 1a). Se componen de interacciones entre evolutivamente conservados motivos que caracterizan a dos familias de proteínas transmembrana integrales de la NE: Sol (sad1 / Unc84) proteínas y Nesprins (Nuclear Sobre SPectRINS). En los mamíferos, Sun1 y Sun2 arproteínas transmembrana e del INM cuyo N-terminal nucleoplasmic región interactúa directamente con A y laminas de tipo B 3-5. En el otro lado del INM, dentro del PNS, las proteínas Sun Harbor un estiramiento-evolutiva conservada de ~ 150 aminoácidos C-terminal llamado el dominio dom Dominios SUN interactúan directamente con la KASH-evolutiva conservada (Klarsicht / ANC-1, Syne homología) de dominio, la firma molecular de Nesprins. KASH dominios consisten en un tramo de ~ 30 aminoácidos C-terminal que sobresale en el PNS seguido por un dominio transmembrana 6. Al menos cuatro genes nesprin distintas (Nesprin1-4) codifican KASH contienen proteínas que se localizan en el NE 7. Las regiones citoplasmáticas de Nesprins, cuyos tamaños varían de ~ 50 kDa (Nesprin4) a un sorprendente 1.000 kDa (gigante Nesprin1), contienen múltiples espectrina repite, así como los motivos específicos que permite su interacción con los componentes del citoesqueleto, tales como actina, plectina y motores moleculares 8- 13. &lt;p class = &quot;jove_content&quot;&gt; Estudios en vertebrados e invertebrados han mostrado que Lamin / Sun / nesprin / motores moleculares constituyen un &quot;eje&quot; conservadas evolutivamente controlar la migración nuclear y anclaje. Varios knock-out (KO) modelos de ratón de componentes complejos LINC se han descrito y fueron fundamentales en la prestación de un marco para comprender las funciones de las proteínas Sol y nesprin en el NE durante el desarrollo de los mamíferos 9,14,15. Sin embargo, estos modelos presentan varios inconvenientes significativos, sobre todo: 1) de dificultad en la interpretación de los fenotipos debido a la celda efectos no autónomos, 2) la dificultad para distinguir las contribuciones fenotípicas de KASH contienen vs. KASH menos nesprin isoformas 16, 3) la redundancia funcional de las proteínas Sun y nesprin en el NE en numerosos tipos de células requiere esquemas de mejoramiento complejos para inactivar todas las interacciones dom-KASH en ratones 17 y 4) la letalidad perinatal de ratones deficientes para el KASH-dominio de ambosNesprins1 y 2 impide el análisis de adultos fenotipos 18. Este protocolo describe un modelo de ratón novedoso diseñado para interrumpir todas las interacciones dom-KASH in vivo, en una célula de manera autónoma y regulado por el desarrollo, evitando así muchos de los inconvenientes descritos anteriormente. Este / modelo de ratón basada en lox Cre se basa en dos conceptos importantes: 1) el dominio KASH de cualquier proteína nesprin conocido es suficiente para dirigir EGFP a la NE en sistemas de cultivo celular y 2) dominios SUN interactuar promiscuamente con dominios Kash, por tanto, la sobreexpresión de cualquier dominio KASH saturará todos los dominios SUN endógenos e inactivar complejos LINC de una manera dominante negativo 17 (Figura 1B). Este protocolo describe la cosecha de tejidos y etapas de procesamiento utilizadas para confirmar la interrupción de todas las interacciones dom-KASH en las células de Purkinje del cerebelo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Gibco</td>
			<td>14200-075</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesion Slides</td>
			<td>StatLab</td>
			<td>M1000W</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>Sigma Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmuEdge Pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Calbindin Antibody</td>
			<td>Sigma Aldrich</td>
			<td>C9848</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-EGFP Antibody</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Nesprin2 Antibody</td>
			<td></td>
			<td></td>
			<td>Previously described in Ref. 21</td>
		</tr>
		<tr>
			<td>Fluorescent Mounting Media</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>2-methyl butane</td>
			<td>Sigma Aldrich</td>
			<td>O3551</td>
			<td></td>
		</tr>
	</tbody>
</table>

validation of a mouse model to disrupt linc complexes in a cell-specific manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Este protocolo ilustra la utilidad de la (/ EGFP-KASH2 CAG-LacZ) modelo de ratón Tg para restringir EGFP-KASH2 expresión a las células de Purkinje del cerebelo utilizando TG (PCP2-CRE) ratones. En Tg (PCP2Cre CAG-EGFP / KASH2) descendencia, el marco de lectura abierto LacZ / V5 se escinde en P6 por la Cre recombinasa lo que conduce a la expresión de EGFP-KASH2 dirigida específicamente a las células de Purkinje (Figura 2A). Como era de esperar, EGFP-KASH2 está dirigido a la envoltura nu...

Watch this Scientific Journal Video about Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner at JoVE.com

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Validación de un modelo de ratón para interrumpir LINC Complejos en una celda de manera específica

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

validation-mouse-model-to-disrupt-linc-complexes-cell-specific

Research

JoVE Journal

Biology

7.3K vistas.  Washington University in St. Louis School of Medicine. Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Video: Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Aplicación de un ensayo de C. elegans la degeneración neuronal de dopamina para la validación del potencial de la enfermedad de Parkinson genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Investigación

Biología

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina Microinyección en pez cebra para modelar la lesión renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-interno celular masiva inyección de células madre embrionarias conduce a mayores tasas de quimerismo

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

Un análisis basado en la citometría de flujo para identificar compuestos que alteran la Unión del ligando de quimiocina CXC marcada con fluorescencia 12 al CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

Un modelo de ratón de la obstrucción Intestinal parcial

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Análisis de espectrometría de masas para identificar la ubiquitylación de CENP-A con etiqueta EYFP (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Un modelo de ratón de inestabilidad lumbar de la columna vertebral

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Adipocitos diferenciados de ratón en cultivo primario: un modelo de resistencia a la insulina

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...