Agradecemos al Prof. Weiguo Zou y A S. Kato por los reactivos y ratones y a los miembros del laboratorio Zou por sus útiles discusiones. También agradecemos al Laboratorio de Estomatología Digitalizada y al Centro de Investigación de Anomalías Craneofaciales del Noveno Hospital Popular de Shanghai por su asistencia. Este trabajo fue apoyado en parte por subvenciones de la Fundación Nacional de Ciencias Naturales de China (NSFC) [81570950,81870740,81800949], Shanghai Summit &Plateau Disciplines, el fondo SHIPM-mu del Instituto de Medicina de Precisión de Shanghai, el Noveno Hospital Popular de Shanghai, la Facultad de Medicina de la Universidad Jiao Tong de Shanghai [JC201809], el Proyecto de Incentivos del Equipo de Innovación de Alto Nivel para la Facultad de Medicina de la Universidad Jiao Tong de Shanghai , el Fondo de Investigación Interdisciplinaria del Noveno Hospital Popular de Shanghai, Facultad de Medicina de la Universidad JiaoTong de Shanghai [JYJC201902]. Y L.J. es un académico de los Talentos Médicos Juveniles Sobresalientes, el Programa de Desarrollo Juvenil "Rising Stars of Medical Talent" de Shanghai y el proyecto "Chen Xing" de la Universidad Jiaotong de Shanghai.

Todos los métodos relacionados con los animales descritos aquí fueron aprobados por el Comité Institucional de Cuidado y Uso de Animales (IACUC) de la Facultad de Medicina de la Universidad Jiaotong de Shanghai.
1. Cría de ratones de deleción Stat3 específicos de osteoclasto
NOTA: Stat3fl /fl ratones se obtuvieron comercialmente. Los ratones Ctsk-Cre fueron proporcionados por S. Kato (Universidad de Tokio, Tokio, Japón12). Los ratones fueron criados y mantenidos bajo condiciones libres de patógenos específicos (SPF) en la instalación animal institucional bajo condiciones estandarizadas.
<ol><li>Emparejar un ratón macho sexualmente maduro con dos ratones hembra de la misma edad. Después de 18 días, revise los controles diarios de recién nacidos. Separe las hembras preñadas y manténgala sola si es necesario. Cambie los ratones machos entre diferentes jaulas de cría si los ratones hembra no estaban preñados dentro de 1 mes del emparejamiento.</li>
	<li>Cruza ratonesfl/fl Stat3a ratones Ctsk-Cre (F0). Recorte las colas para el genotipado y mantenga el macho Stat3fl /+; Ratones Ctsk-Cre hasta que son sexualmente maduros, que es alrededor de 6 semanas de edad (F1). Utilice los siguientes cebadores: Stat3 F-TTGACCTGTGCTCCTACAAAAA; Stat3 R-CCCTAGATTAGGCCAGCACA; Ctsk-cre F-GAACGCACTGATTTCGACCA; Ctsk-cre R-GCTAACCAGCGTTTTCGTTC.</li>
	<li>Cruz 6-semana de edad macho Stat3fl /+; Ratones Ctsk-Cre con hembras de ratones Stat3fl/fl. Recorte las colas para el genotipado y mantenga el macho Stat3 fl / fl; Ratones Ctsk-Cre hasta que son sexualmente maduros, que es alrededor de 6 semanas de edad (F2).</li>
	<li>Cruz 6-semana de edad macho Stat3 fl/fl; Ratones Ctsk-Cre con ratones hembras Stat3fl/fl (F3). Recorte las colas para el genotipado y reemplace los ratones reproductores viejos con ratones más jóvenes a tiempo (F3 + N).</li>
</ol>2. Recolección de especímenes
<ol><li>Eutanasia seis pares de ratones machos de 8 semanas de edad Stat3fl/fl y Stat3Ctsk littermate por separado con asfixia de dióxido de carbono. NOTA: El caudal de CO2 desplaza el 30% del volumen de la jaula por minuto (por ejemplo, para una jaula de 45 cm x 30 cm x 30 cm, la tasa de flujo de CO2 es de 40 L/min).</li>
	<li>Coloque los ratones en decúbito supino. Dislocar las articulaciones bilaterales de la cadera suavemente con la mano. Use tijeras oftálmicas para cortar la piel verticalmente de la tibia distal y luego diseccione toda la piel de la extremidad posterior.</li>
	<li>Corte el ligamento articular de la articulación de la cadera derecha y la articulación de la rodilla con tijeras para separar la extremidad posterior. Corte el trocánter y la unión del peroné y luego sumerja el miembro posterior en paraformadehído al 4%. Mantenga las extremidades posteriores derechas para el paso 3. Corte adecuadamente el hueso en ambos extremos para sumergir completamente y fijar la médula ósea con paraformadehído al 4%.</li>
	<li>Corte el ligamento articular de la articulación izquierda de la cadera y la articulación de la rodilla con tijeras, retire suavemente el tejido blando y separe cuidadosamente la tibia y el fémur. Sumergir la tibia y el fémur por separado en etanol al 75%. Mantenga los fémures para el paso 4 y las tibias para el paso 6. Asegúrese de mantener el trocánter intacto.</li>
</ol>3. Preparación de la sección de parafina
<ol><li>Fijar el miembro posterior derecho en paraformadehído al 4% a 4 °C durante 48 h.</li>
	<li>Descalcificar: Lavar suavemente las muestras con 1x PBS durante 10 min 3 veces. Descalcificar las muestras en EDTA al 15% (150 g EDTA en 800 mL de ddH2O y 100 mL de PBS 10x) con un descalcificador ultrasónico durante 3 a 4 semanas hasta que los huesos puedan ser doblados. Reemplácelo con líquido descalcificante fresco cada dos días.</li>
	<li>Lave suavemente las muestras 3x con 1x PBS y luego sumérjalas en etanol al 75% a 4 °C durante la noche.</li>
	<li>Deshidratar: En el segundo día, sumergir secuencialmente las muestras en etanol al 95%, etanol al 100% y xileno, cada una durante 1 h dos veces.</li>
	<li>Sumergir los especímenes en 1/2 xileno 1/2 parafina durante 30 min. Sumergir los ejemplares en parafina a 65 °C durante la noche.</li>
	<li>Incrustar: Seleccione un tanque de incrustación adecuado para la incrustación. Coloque la tibia uniformemente debajo. Coloque el fémur y la tibia en un ángulo de 90°. Después de que la parafina se haya enfriado y solidificado por completo, retírela del tanque de incrustación. Numerar los especímenes y almacenarlos a -20 °C durante la noche.</li>
	<li>Cortar secciones de 5 μm de espesor continuamente usando el microtomo. Corte de 20 a 40 secciones. Extienda las secciones en agua de 37 °C, adhiérelas a los portaobjetos del microscopio y hornee a 42 °C durante la noche.</li>
</ol>4. Exploración y análisis de micro-TC
<ol><li>Escanee los fémures izquierdos con un escáner micro-CT. Resolución: 10 μm; Voltaje: 70 kV; Corriente: 114 μA; Fliter: 0.5 mm Al; Paso de rotación: 0.5°.</li>
	<li>Reconstruya imágenes 3D del hueso cortical y del hueso trabecular utilizando el software de soporte del escáner siguiendo las instrucciones del fabricante. Los ROIs están en un ancho total de 1 mm de hueso trabecular cerca de la placa de crecimiento distal y en una sección total de 1 mm de ancho de hueso cortical en el centro de los fémures.</li>
	<li>Calcule los parámetros cuantitativos de la microarquitectura: densidad mineral ósea (DMO), fracción de volumen óseo (BV/TV), espesor trabecular (Tb.Th.), número trabecular (Tb.N.), separación trabecular (Tb.Sp.) y grosor óseo cortical (Ct.Th.).</li>
</ol>5. Tinción trap
<ol><li>Hornear las secciones de parafina a 65 °C durante 30 min.</li>
	<li>Dewax: Sumergir las secciones en xileno durante 10 min. Realice este paso 3x con xileno fresco.</li>
	<li>Rehidratar: Sumergir las secciones secuencialmente en etanol al 100%, etanol al 95%, etanol al 70% y ddH2O, cada una durante 5 min dos veces.</li>
	<li>Prepare la solución de tinción utilizando el kit de tinción TRAP siguiendo las instrucciones del fabricante y caliente a 37 °C. NOTA: La solución de tinción trap debe prepararse recientemente inmediatamente antes de cada ensayo.</li>
	<li>Añadir una solución de tinción de 50–100 μL a cada muestra e incubar en una cámara húmeda de 37 °C durante 20–30 min. Compruebe el estado de tinción de los osteoclastos bajo un microscopio de luz cada 5 minutos hasta que se puedan ver osteoclastos multinucleados rojos. Finalizar la reacción con ddH2O.</li>
	<li>Contratendía en solución de hematoxilina durante 30 s. Crear un color azul estable por inmersión en 1% solución de amoníaco durante 1 min. Enjuague en agua del grifo que corre lentamente.</li>
	<li>Monte las secciones con cubrebocas usando bálsamo neutro y seco durante la noche.</li>
	<li>Captura de 3 a 5 campos de interés mediante un microscopio. Analice el perímetro trabecular por la Imagen J: mida la longitud de la barra de escala (Ls) usando la herramienta 'línea recta' como L1, luego mida la longitud del perímetro trabecular usando la herramienta 'línea segmentada' como L2, la longitud física (Lp)= Ls*L2 /L1). Cuente el número de células TRAP-positivas con más de tres núcleos.</li>
</ol>6. Calein y alizarin rojo doble etiquetado
<ol><li>Preparación de la muestra: Inyecte por vía intraperitoneal 20 mg/kg de caleína (1 mg/mL en solución de NaHCO3 al 2%) el día 0, y 25 mg/kg de alizarina roja S (AL, 2 mg/mL en H2O) el día 4. Sacrificar ratones el día 7. Desasocie cuidadosamente las tibias y fije en paraformadehído al 4% a 4 °C durante 48 h.</li>
	<li>Deshidratar: Después de la fijación, lavar suavemente las tibias 3x con 1x PBS. Sumergir secuencialmente las muestras en etanol al 95%, etanol al 100% y xileno durante 5 min dos veces por separado.</li>
	<li>Sumergir las muestras en acetona durante 12 h, en 1/2 acetona 1/2 resina durante 2 h, y en resina pura en un horno de secado durante la noche. NOTA: La resina fue preparada por resina disponible comercialmente (por ejemplo, Embed 812 Resin) de acuerdo con las instrucciones del fabricante.</li>
	<li>Incrustar: Agregue resina pura en un tanque de incrustación de gel de sílice adecuado y coloque las muestras suavemente para evitar burbujas. Polimerizar la resina en un horno de secado a 60 °C durante 48 h.</li>
	<li>Corte las muestras en secciones de 5 μm de espesor continuamente con un microtomo rotatorio. Guarde el resto de las muestras con desecante a temperatura ambiente.</li>
	<li>Adherir las secciones con pinzas en una gota de 75% de alcohol. Monte las secciones con cubrebocas usando bálsamo neutro. Capture el etiquetado de fluorescencia rojo y verde con un microscopio de fluorescencia.</li>
	<li>Mida el ancho entre dos líneas de etiquetado (Ir.L.Wi), perímetro trabecular de etiqueta simple (sL.Pm), perímetro trabecular de doble etiqueta (dL.Pm) y perímetro trabecular total (Tb.Pm). Calcular la tasa de aposición mineral (MAR) y la tasa de formación ósea (BFR/BS). MAR = Ir.L.Wi/días de intervalo. BFR/BS= (dL.Pm±sL.Pm/2)*MAR/Tb.Pm*100 %.</li>
</ol>

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Los autores no tienen nada que revelar.

Los modelos de ratón genéticamente modificados se utilizan comúnmente para estudiar el mecanismo y el tratamiento farmacéutico de las enfermedades humanas13. Los ratones Ctsk-Cre han sido ampliamente utilizados para estudios funcionales de osteoclastos6. El actual estudio describió los protocolos de los métodos para analizar fenotipo esquelético y para estudiar los genes críticos que controlaban actividad osteoclasta in vivo.
El ...

El hueso esquelético es el principal órgano de carga del cuerpo humano y está bajo la presión del entorno interno y externo durante la marcha y el ejercicio1. A lo largo de la vida, los huesos pasan continuamente por la auto-renovación, que es equilibrada por osteoblastos y osteoclastos. El proceso de los osteoclastos que limpian los huesos viejos y los osteoblastos que forman el hueso nuevo mantiene la homeostasis y la función mecánica del sistema esquelético2. La alteración en el equilibrio puede inducir enfermedades metabólicas óseas, como la osteoporosis. La osteoporosis, que es causada por el exceso de actividad osteoclástica, es prevalente a nivel mundial y causa pérdidas económicas sustanciales a la sociedad2,3,4. De acuerdo con el número limitado de fármacos disponibles para el tratamiento de la osteoporosis y su riesgo de efectos adversos4,es importante desvelar los detalles de la formación y actividad de los osteoclastos.
Los osteoclastos derivados del linaje hematopoyético monocitos/macrófagos tienen múltiples núcleos (pueden tener de 2 a 50 núcleos) y son grandes (generalmente mayores de 100 μm de diámetro)2. Aunque la exploración de mecanismos y la investigación de las drogas para los desordenes osteoclastic se hayan mejorado extensamente vía cultura osteoclasta in vitro, las reacciones orgánicas complicadas hacen in vivo la evidencia indispensable para la terapia apuntada. Debido a las similitudes genéticas y fisiopatológicas entre ratones y humanos, los modelos de ratón genéticamente modificados se utilizan comúnmente para estudiar los mecanismos y los tratamientos farmacéuticos de las enfermedades humanas in vivo6. El sistema Cre-loxP es una tecnología ampliamente utilizada para la edición de genes de ratón y ha permitido a los investigadores investigar las funciones de los genes de una manera específica de tejidos/células5. La catepsina K (CSTK) es una cisteína proteasa secretada por osteoclastos que puede degradar el colágenoóseo 8. Está bien aceptado que CTSK se expresa selectivamente en osteoclastos maduros; por lo tanto, los ratones Ctsk-Cre son considerados como una herramienta útil para estudios funcionales de osteoclastos y se ha utilizado6.
La familia del transductor de señales y activador de la transcripción (STAT) es clásica y altamente significativa en la inmunidad y en la progresión y desarrollo del cáncer7,8. Entre siete ESTAT, STAT3 se divulga para ser el más relevante al homeostasis del hueso9,10. Varios estudios in vivo han reportado que la inactivación específica de STAT3 en osteoblastos disminuye la formaciónósea 9,10. Sin embargo, la evidencia sólida con respecto a la participación de STAT3 en la formación de osteoclastos y el metabolismo óseo in vivo todavía es limitada. Recientemente, proporcionamos evidencia in vivo con un modelo de ratón de deleción Stat3 osteoclasta específico(Stat3fl / fl; Ctsk-Cre, en adelante llamado Stat3Ctsk) que STAT3 participa en la diferenciación osteoclasta y metabolismo óseo11. En el presente estudio, describimos los métodos y protocolos que utilizamos para analizar los cambios en la masa ósea, la histomorfología ósea y el anabolismo óseo y el catabolismo de los ratones Stat3Ctsk con el fin de estudiar la influencia de la deleción osteoclasta-específica de STAT3 en la homeostasis ósea.

<table>
	<tbody>
		<tr>
			<td>4% Paraformaldehyde solution</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>E672002</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>80000360</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonia solution</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Ctsk-Cre mice</td>
			<td></td>
			<td></td>
			<td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td>
		</tr>
		<tr>
			<td>DDSA</td>
			<td>Electron Microscopy Sciences</td>
			<td>13710</td>
			<td></td>
		</tr>
		<tr>
			<td>DeCa RapidlyDecalcifier</td>
			<td>Pro-Cure</td>
			<td>DX1100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP-30</td>
			<td>Electron Microscopy Sciences</td>
			<td>13600</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>EMBED 812 RESIN</td>
			<td>Electron Microscopy Sciences</td>
			<td>14900</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescence microscope</td>
			<td>Olympus</td>
			<td>IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin solution</td>
			<td>Beyotime Biotechanology</td>
			<td>C0107</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-CT</td>
			<td>Scanco Medical AG</td>
			<td>&#956;CT 80</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>10018918</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>E675007</td>
			<td></td>
		</tr>
		<tr>
			<td>NMA</td>
			<td>Electron Microscopy Sciences</td>
			<td>19000</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>A601889</td>
			<td></td>
		</tr>
		<tr>
			<td>rotary microtome</td>
			<td>Leica</td>
			<td>RM2265</td>
			<td></td>
		</tr>
		<tr>
			<td>Stat3fl/fl mice</td>
			<td>GemPharmatech Co., Ltd</td>
			<td>D000527</td>
			<td></td>
		</tr>
		<tr>
			<td>TRAP staining kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene</td>
			<td>Shanghai Experimental Reagent Co., Ltd.</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

skeletal phenotype analysis of a conditional stat3 deletion mouse model

Los modelos transgénicos del ratón son potentes para entender los genes críticos que controlan la diferenciación y la actividad del osteoclast, y para estudiar mecanismos y tratamientos farmacéuticos de la osteoporosis. Los ratones de catepsina K (Ctsk)-Cre han sido ampliamente utilizados para estudios funcionales de osteoclastos. El transductor de señales y activador de la transcripción 3 (STAT3) es relevante en la homeostasis ósea, pero su papel en los osteoclastos in vivo sigue estando mal definido. Para proporcionar la evidencia in vivo de que STAT3 participa en la diferenciación de osteoclastos y el metabolismo óseo, se generó un modelo de ratón de deleción Stat3 osteoclasta específico(Stat3 fl / fl; Ctsk-Cre) y analizó su fenotipo esquelético. La exploración de Micro-CT y la reconstrucción 3D implicaron la masa creciente del hueso en los ratones condicionales del golpe de gracia. La coloración de H&E, la coloración doble roja de la caleína y del alizarin, y la coloración ácida tartrato-resistente de la fosfatasa (TRAMPA) fueron realizadas para detectar metabolismo del hueso. En definitiva, este protocolo describe algunos métodos y técnicas canónicas para analizar el fenotipo esquelético y estudiar los genes críticos que controlan la actividad osteoclasta in vivo.

Usando el actual protocolo, los ratones específicos osteoclast de la canceladura Stat3 fueron generados para estudiar la influencia de la canceladura STAT3 en la diferenciación osteoclasta. Los ratones Stat3Ctsk y sus compañeros de camada de tipo salvaje (WT) fueron criados y mantenidos después del genotipado. Los macrófagos de médula ósea se aislaron y cultivaron en osteoclastos, y se demostró la deleción de STAT3en ratones Stat3Ctsk <strong cla...

Watch this Scientific Journal Video about Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model at JoVE.com

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Este protocolo describe un método canónico para entender los genes críticos que controlan la actividad osteoclasta in vivo. Este método utiliza un modelo de ratón transgénico y algunas técnicas canónicas para analizar el fenotipo esquelético.

Análisis de fenotipo esquelético de un modelo de ratón de eliminación condicional de Stat3

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

skeletal-phenotype-analysis-conditional-stat3-deletion-mouse

Research

JoVE Journal

Biology

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

4.3K vistas.  National Clinical Research center of Stomatology. Este protocolo describe un método canónico para entender los genes críticos que controlan la actividad osteoclasta in vivo. Este método utiliza un modelo de ratón transgénico y algunas técnicas canónicas para analizar el fenotipo esquelético.

Video: Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Investigación

Biología

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Análisis de la elaboración de un fenotipo morfológico en función de la concentración de proteínas en la levadura en ciernes

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

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 ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Culturas alternativas para Pluripotentes Stem Cell Production, mantenimiento y análisis genético

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Detección y análisis de daño del ADN en el ratón músculo esquelético<em&gt; In Situ</em&gt; Con el método TUNEL

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

Cryosectioning de regiones contiguas de un músculo esquelético solo ratón para la expresión de genes y análisis histológicos

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

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 ...

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 ...