Los autores desean agradecer al Dr. Rizaldy Scott en el Hospital Mount Sinai en Toronto, Ontario por su don de la primaria Rac1 fibroblastos flox / flox de embriones de ratón. Este trabajo fue apoyado por subvenciones de funcionamiento para NJ de los Institutos Canadienses de Investigación en Salud (CIHR, 2009-93.526 MOP) y el de Ciencias Naturales e Ingeniería de Investigación de Canadá (NSERC, RG 327372 a 06). SH y MW son apoyados por CGSMA y M-CGS premios de CIHR y NSERC, respectivamente. Steve Hawley Testamentos y Melanie contribuyeron igualmente a este trabajo.

Las células de descongelación <ol><li> Obtener los fibroblastos congelados embriones de ratón (MEFs) (generado previamente del cultivo de embriones primarios individuales) a partir de nitrógeno líquido. </li><li> Descongelar las células mediante la inmersión en el vial a 37 ° C con agitación hasta que se hayan descongelado por completo. </li><li> Añadir 9 ml de DMEM que se ha complementado con suero fetal bovino al 10% y 1% de penicilina-estreptomicina a una placa de cultivo celular de 10 cm. </li><li> Añadir la suspensión de células descongeladas, gota a gota a la placa, el rock suavemente la placa para dispersar las células de la placa y refrigerarla en un 37 ° C CO, 5% 2 incubadora durante la noche. Tenga en cuenta que si las células son sensibles a DMSO (del proceso de congelación), los medios de comunicación inicial puede ser sustituido por medio fresco después que las células se han adherido a la placa (aproximadamente cuatro horas después de la chapa). También tenga en cuenta que la confluencia de las células después de la descongelación dependerá del número de células congeladas, la tasa de recuperación de las células después de la congelación, y la tasa de crecimiento de las células. </li></ol> Las células de los pases <ol start="5"><li> Cuando las células han llegado a prácticamente el 100% de confluencia (por ejemplo: al día siguiente), aspirado de los medios de comunicación y lavar las células con 5 ml de PBS estéril. </li><li> Quitar PBS y añadir 1 ml de tripsina que contiene 10 mM EDTA y la placa de lugar en la incubadora durante unos 5 minutos para permitir que las células se desprenden de la placa. </li><li> Cuando las células se han desprendido, agregar 9 ml de los medios de comunicación a las células con tripsina, pipeta de 3.2 veces para dispersar a grupos, añadir 1 ml de esta suspensión, gota a gota en un plato nuevo que contiene 9 ml de los medios de comunicación, y este lugar en los 37 ° C incubadora durante la noche . La presencia de FBS en los medios de inactivar la tripsina, alternativamente, las células se pueden lavar primero a diluir la tripsina. </li></ol> Conteo de células y placas en la cubierta se desliza <ol start="8"><li> Eliminar las células de la incubadora, lavar y trypsinize como antes, excepto que esta vez añadir 5-6 ml medios de comunicación de nuevo a las células con tripsina en lugar de 9 ml. </li><li> Las células con una pipeta a fondo para asegurar la disociación completa en una única suspensión de células, y extraer 15μL para combinar con 15μL de azul tripán en un tubo de microcentrífuga separados. </li><li> Mezclar esta suspensión celular / azul tripán pipeteando arriba y abajo varias veces, y añadir 15μL del hemocitómetro. </li><li> Bajo el microscopio, las células muertas que aparecen en azul. Cuente el número de células claras / incoloro (que viven) en una de las redes de 4X4 en el hemocitómetro. Repita este cuenta con las otras tres redes de 4X4 (denominado A, B, C y D) y el uso de la siguiente ecuación para calcular el número de células por microlitro de la suspensión: <img src="/files/ftp_upload/2160/2160eq0.jpg" alt="La ecuación 1" /></li><li> A través de la optimización del protocolo, hemos determinado que el recubrimiento 30.000 resultados células de la densidad celular apropiada para la visualización a la conclusión del experimento. </li><li> Para calcular el volumen de suspensión celular necesario para 30.000 celdas, utilice la siguiente ecuación: <img src="/files/ftp_upload/2160/2160eq1.jpg" alt="La ecuación 2" /></li><li> A continuación, coloque una hoja de cubierta de vidrio estéril en cada pocillo de una placa de seis bien y añadir 2 ml de los medios de comunicación a cada pocillo, y luego añadir, gota a gota de la suspensión de su celular el volumen requerido por 30.000 células en cada pocillo. Tenga en cuenta que se desliza cubierta de vidrio se utilizan para este experimento que vamos a visualizar las células mediante microscopía de fluorescencia en un momento posterior, no todas las aplicaciones necesariamente requerirá de la microscopía, y por lo tanto la cubierta se desliza puede no ser necesaria. </li></ol> Virus de transducción <ol start="15"><li> Después de varias horas o (tiempo suficiente para asegurar que las células se adhieren a la cubierta se desliza) durante la noche, retire el material y lavar las células con 1 ml de suero libre de DMEM. </li><li> Añadir 1 ml de medio libre de suero a cada pocillo de la adición de los virus. Para este experimento, adeno-Cre virus se obtuvo comercialmente de Biolabs Vector. </li><li> Optimización de protocolos anteriores han demostrado que una multiplicidad de infección (MOI), de 500 proporciona transducción de genes de alta eficiencia en la atención primaria MEFs con poco o ningún efecto sobre la viabilidad celular. MOI se puede calcular de la siguiente manera: <img src="/files/ftp_upload/2160/2160eq2.jpg" alt="La ecuación 3" /> <img src="/files/ftp_upload/2160/2160eq3.jpg" alt="La ecuación 4" /></li><li> Agregar el volumen calculado de virus directamente a cada uno, así que va a ser infectados, agite suavemente la placa para dispersar a los virus de las células y el lugar en la incubadora a 37 ° C durante la noche. </li><li> A la mañana siguiente, eliminar el virus que contienen los medios de comunicación de las células y lavar las células en 1 ml de PBS estéril y luego agregar 2 ml de los medios de comunicación regulares para cada pozo. </li><li> En el caso de las células de Rac1 se ejemplifica en este experimento, se produjeron cambios morfológicos en las células en aproximadamente 72 horas después de transducción. </li></ol> Resultados representante Las células se visualizand mediante la tinción con el colorante phalloidin actina (a efectos de imágenes solamente) y la imagen con el Leica TCS-SP5 multifotónica microscopio láser confocal de barrido en el Centro de Análisis Avanzados de la Universidad de Guelph. La figura 1 muestra la morfología alargada y contratados de la Rac1 flox / flox células que fueron expuestos a virus adeno-Cre en comparación con las mismas células no expuestas al virus. <img src="/files/ftp_upload/2160/2160fig1.jpg" alt="Figura 1" /> Figura 1. Comparación entre Rac1 flox / flox células infectadas con virus adeno-Cre (izquierda) en comparación con las células no infectadas de la misma fuente (derecha).

<ol>
	<li>Guo, F., Debidda, M., Yang, L., Williams, D. A., Zheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Deletion+of+Rac1+GTPase+Reveals+Its+Critical+Role+in+Actin+Stress+Fiber+Formation+and+Focal+Adhesion+Complex+Assembly.">Genetic Deletion of Rac1 GTPase Reveals Its Critical Role in Actin Stress Fiber Formation and Focal Adhesion Complex Assembly.</a> J. Biol. Chem. 281, 18652-18659 (2006).</li><li>Glogauer, M., Marchal, C. C., Zhu, F., Worku, A., Clausen, B. E., Foerster, I., Marks, P., GP, D. o. w. n. e. y., Dinauer, M., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1+deletion+in+mouse+neutrophils+has+selective+effects+on+neutrophil+functions.">Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions.</a> J. Immunol. 170, 5652-5657 (2003).</li><li>Prost, S., Sheahan, S., Rannie, D., Harrison, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+Cre+deletion+of+floxed+sequences+in+primary+mouse+cells+is+an+efficient+alternative+for+studies+of+gene+deletion.">Adenovirus-mediated Cre deletion of floxed sequences in primary mouse cells is an efficient alternative for studies of gene deletion.</a> Nucleic Acids Res. 29, E80-E80 (2001).</li><li>Rijnkels, M., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-Cre-mediated+recombination+in+mammary+epithelial+early+progenitor+cells.">Adenovirus-Cre-mediated recombination in mammary epithelial early progenitor cells.</a> J. Cell. Sci. 114, 3147-3153 (2001).</li><li>Vidali, L., Chen, F., Cicchetti, G., Ohta, Y., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1-null+mouse+embryonic+fibroblasts+are+motile+and+respond+to+platelet-derived+growth+factor.">Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor.</a> Mol. Biol. Cell. 17, 2377-2390 (2006).</li><li>Wang, H., Xie, H., Zhang, H., Das, S. K., Dey, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+recombination+by+adenovirus-driven+Cre+in+the+mouse+uterus.">Conditional gene recombination by adenovirus-driven Cre in the mouse uterus.</a> Genesis. 44, 51-56 (2006).</li></ol>

Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por el Consejo Canadiense de los Animales.

El video que acompaña un ejemplo de cómo un virus recombinante se puede utilizar para extirpar un gen específico in vitro utilizando la recombinasa Cre. El desarrollo de este protocolo sin embargo, puso de manifiesto algunos puntos en particular vale la pena abordar. <ul><li> Los procedimientos de seguridad siempre se debe emplear cuando se trabaja con adenovirus. Una bata de laboratorio y se deben usar guantes en todo momento mientras trabaja con el virus. Los medios de comunicación y artículos de plástico deben ser tratados con lejía al 10% por lo menos durante 30 minutos, y utensilios de plástico deben ser esterilizados en autoclave a partir de entonces. </li><li> Evitar la repetición de congelación-descongelación del stock de virus ya que esto puede afectar a título de virus y por lo tanto reducen la eficiencia de transducción. Pequeñas cantidades de stock de virus debe ser, inicialmente alícuotas en tubos separados. </li><li> MOI que van desde 50 hasta 500 fueron probados durante el desarrollo del método y las tasas de eficiencia de transducción se observaron en la mayoría de los casos, sin efectos negativos sobre la viabilidad celular. </li><li> Adeno-GFP se utilizó también durante el desarrollo de métodos para detectar rápidamente la eficiencia de transducción. Este control (o un vector vacío) debe llevarse a cabo durante el experimento para asegurarse de que la infección viral por sí sola no resultará en cambios celulares. Del mismo modo, si la escisión del gen floxed de interés en sus células no se traduce en cambios celulares (es decir, cambios en la morfología o la viabilidad), y luego infectan el mismo tipo de células con virus adeno-GFP es un control importante utilizar para demostrar que la transducción de virus ocurriendo. </li></ul> Más allá del examen de las cascadas de señalización en la cultura, el enfoque de adeno-Cre también se ha empleado en vivo para eliminar condicionalmente genes de animales de experimentación 3,4, y para marcar poblaciones específicas de células de un organismo 6. El sistema de adenovirus también puede ser adaptado para introducir otros genes de la recombinasa Cre en las células huésped. Dos principales ventajas de la utilización de este vector de entrega son sus transducción de alta eficiencia y la entrega de genes, y la falta de integración con el ADN del huésped. Sin embargo, la generación de nuevos adenovirus recombinante puede ser mano de obra intensiva. El protocolo descrito aquí por lo tanto, aprovecha la potencia del sistema adenoviral mediante la explotación de los desarrollados anteriormente adeno-Cre virus, y el acoplamiento que con nuestro alelo floxed de interés, Rac1. A través de esta llamada condicional a cabo el experimento, hemos confirmado que la transducción con el virus adeno-Cre de MEFs que lleva un alelo Rac1 floxed en verdad causa la escisión de Rac1 que conducen a los cambios en la morfología celular característica de las células deficientes en Rac1 2,5.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ad-CMV-Cre virus</td>
<td>&nbsp;</td>
<td>Vector Biolabs</td>
<td>1045</td>
<td>&nbsp;</td>
<tr>
<td>DME High glucose media 500mL</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SH3002201</td>
<td>&nbsp;</td>
<tr>
<td>FBS Canadian origin 500mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CAA15-701</td>
<td>&nbsp;</td>
<tr>
<td>PEN-STREP 1X solution 100mL</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SV30010</td>
<td>&nbsp;</td>
<tr>
<td>Texas Red-X phalloidin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>T7471</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T4549</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

adenovirus-mediated genetic removal of signaling molecules in cultured primary mouse embryonic fibroblasts

La capacidad de eliminar genéticamente los componentes específicos de las diversas cascadas de señalización celular ha sido una herramienta fundamental en el análisis moderno de transducción de señales. Un método particular para lograr la supresión condicional es a través de la utilización del sistema Cre-loxP. Este método consiste en que flanquean el gen de interés con los sitios loxP, que son secuencias específicas de reconocimiento para la proteína recombinasa Cre. La exposición de los llamados floxed (flanqueado por sitios loxP) del ADN de esta enzima provoca un evento de recombinación Cre-mediada en los sitios loxP, y la escisión posterior de los genes que intervienen 3. Diferentes métodos existentes para administrar la recombinasa Cre en el sitio de interés. En este vídeo, que muestran el uso de un adenovirus que contiene el gen de la recombinasa Cre para infectar fibroblastos primarios de embriones de ratón (MEFs) obtenidas de embriones que contienen una floxed Rac1 alelo 1. Nuestra razón de ser para la selección de Rac1 MEFs para nuestros experimentos es que los cambios morfológicos claros se puede ver a la supresión de Rac1, debido a alteraciones en el citoesqueleto de actina a 2,5. 72 horas después de la transducción viral y la expresión de Cre, las células fueron teñidas con tinte phalloidin la actina y la imagen por microscopía confocal de barrido láser. Se observó que MEFs que habían estado expuestos al virus adeno-Cre aparecieron contratados y en la morfología alargada en comparación con las células no infectadas, de acuerdo con los informes anteriores 2,5. El método de adenovirus Cre recombinasa entrega es una ventaja ya que el virus adeno-Cre está fácilmente disponible, y la supresión de genes a través de Cre en casi el 100% de las células se puede lograr con la infección por adenovirus optimizado.

Watch this Scientific Journal Video about Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts at JoVE.com

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

En este vídeo se utiliza un adenovirus que porta el gen de la recombinasa Cre para infectar fibroblastos primarios de embriones de ratón que lleva un alelo floxed Rac1.

Mediada por adenovirus eliminación de moléculas de señalización genética en cultivos de fibroblastos de embriones de ratón primaria

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

adenovirus-mediated-genetic-removal-signaling-molecules-cultured

Research

JoVE Journal

Biology

24.8K vistas.  University of Guelph. En este vídeo se utiliza un adenovirus que porta el gen de la recombinasa Cre para infectar fibroblastos primarios de embriones de ratón que lleva un alelo floxed Rac1.

Video: Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Imágenes en vivo del denso núcleo de vesículas en la Primaria neuronas del hipocampo cultivadas

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Investigación

Biología

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Generación de células madre pluripotentes inducidas por reprogramación de fibroblastos embrionarios de ratón con un factor de transcripción Cuatro, doxiciclina inducible sistema de transducción de lentivirales

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons2. This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate3,4 and the chemoattractive activities of Netrin15 and Sonic Hedgehog (Shh)6 in the floor plate in the spinal cord.
Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels7. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag18. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons3,4 as well the necessity for this signal to direct the commissural trajectory9.

This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.

Ensayo de la capacidad de difusible moléculas de señalización para reorientar embrionarias axones de la comisura espinal

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

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 ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

La eliminación rápida de fibroblastos de alta densidad Humanos culturas de células madre embrionarias

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

El análisis genético rápido de epitelio-mesenquimal de señalización durante la regeneración del cabello

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

Orgánulos Transporte en Cultivadas<em&gt; Drosophila</em&gt; Celdas: S2 Línea Celular y neuronas primarias.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Medición de la Absorción de Glucosa y Respuesta a la Estimulación de la Insulina en<em&gt; In Vitro</em&gt; Myotubes primario humano cultivado

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

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 ...