Este protocolo se basa en las técnicas aprendidas en el laboratorio de Francis-André Wollman en el Instituto de Francia Biologie físico-Chimique, París,. Los autores tienen una deuda de gratitud a Jacqueline Girard-Bascou, que originalmente presentó a las técnicas de estimulación cerebral profunda Chlamydomonas genética. Chlamydomonas la investigación en laboratorio de los autores con el apoyo de la Fundación Nacional de Ciencias premio MCB-0646350.

Parte 1: Preparación de las cepas de la gametogénesis Nota: Todas las manipulaciones deben llevarse a cabo a temperatura ambiente. Temperaturas altas o más bajas que afectan a la velocidad de germinación cigoto y puede afectar negativamente a la viabilidad. Consideración importante: Es esencial que las cepas que se aparearon están creciendo vigorosamente y están libres de contaminación de hongos o bacterias. Si las tensiones no han sido transferidos con regularidad, el paso en medio rico debe preceder a la generación de gametos. <ol><li> Las cepas que se aparearon se transfieren a placas TAP (100 x 15 mm), 1-2 cepas por placa. Las células deben ser cultivadas en una franja concentrada alrededor de 1 cm de ancho. </li><li> Incubación en placas TAP debe continuar hasta que una capa gruesa de células de crecimiento robusto está presente, con transmisiones intermedias, si es necesario. Las cepas que no han sido recientemente pasajes pueden requerir un pasaje y un total de 1 semana, mientras que las cepas salvajes en el uso regular sólo puede tomar unos pocos días y los pasajes no. Mutantes que son sensibles a la luz puede requerir más tiempo. </li><li> La transferencia de todas las células de las placas TAP a las placas N10, que son deficientes de nitrógeno y comenzará a inducir la gametogénesis. Transferencia de las células como una losa de espesor, todo lo que estuvieron presentes en la placa de TAP. Esto facilitará la posterior recogida de las células. Después de 3 días en N10, las células están dispuestas a someterse a la gametogénesis. La división celular es lento en la N10, sin embargo, las células deben permanecer de color verde oscuro. Cualquier cepa contaminada debe ser descartado como cigotos viables no se obtendrá. </li></ol> Parte 2: El apareamiento <ol><li> Cada cepa se transfiere a un matraz Erlenmeyer de 50 ml y se resuspendió en aproximadamente 2,5 ml de agua destilada estéril. Esto se puede hacer ya sea usando un asa de alambre o un palo estéril, lo importante es que las células están bien baja y no en grupos. El volumen exacto de agua debe ser ajustada de manera que la concentración celular, medido por los ojos, es casi el mismo para cada cepa. </li><li> Las células se agitan bajo una luz intensa (1.260 E μ / m 2 / s) por 2 horas, tiempo durante el cual las células se convertirán en los gametos móviles. Motilidad debe evaluarse bajo un microscopio de luz. Si los gametos son la natación mal, el apareamiento es probable que sea ineficaz. Sin embargo, incluso mal cepas móviles se aparea con una frecuencia reducida. </li><li> Volúmenes que viene, igual de mt + y mt-gametos se combinan en un matraz aforado de solo 50. Los gametos se dejan bajo una luz intensa (1.260 E μ / m 2 / s) sin agitación. </li><li> El apareamiento se puede comprobar de inmediato bajo el microscopio de luz. Pares de gametos se forman rápidamente, y aparecen como células de rápido movimiento se unió a los flagelos. Si el apareamiento parece ser muy rápido, las muestras deben ser tomadas dentro de los 30 minutos por hora -1 para la generación de cigotos. Si no, el apareamiento se puede comprobar cada hora, aunque esto es opcional. Desde los tiempos de dar la densidad óptima de los cigotos se varían ampliamente entre los cruces, lo mejor es tomar alícuotas de la mezcla de apareamiento en momentos diferentes después de la combinación de los gametos, por ejemplo, después de 1 hora, 2 horas, 3 horas y 4 hrs. Esto se hace mediante la transferencia de 300 muestras de l a TAP-3% de agar Difco 60 x 15 mm placas, manchas 04.03 por placa. Tenga cuidado de no agitar la mezcla de apareamiento, ya que puede alterar parejas de apareamiento. Agar Difco se utiliza porque tiene menos contaminantes que los de menor precio agar, y será más fácil ver los cigotos en el microscopio. La mezcla de apareamiento no utilizados pueden ser dejados en el matraz durante la noche, y la calidad de la reacción de acoplamiento será evidente al día siguiente. Cuando el apareamiento es eficiente, los cigotos se adhiere a la pared o el fondo del recipiente y el medio se parece clara. Incluso la agitación del matraz no podrá desalojar a los cigotos. Si nada se pega al vidrio cuando se agita con fuerza razonable, entonces el apareamiento era ineficiente o no tener lugar. </li><li> Las placas TAP Difco mezcla que contiene el apareamiento se secan en una campana estéril, con las tapas entreabiertas, hasta que el líquido se haya absorbido la superficie. Las placas normalmente se dejan en la luz (700 NE / m 2 / s) durante 18 horas, luego se envuelve en papel de aluminio, ya que la maduración zigospora sólo se llevará a cabo en la oscuridad. Las cepas cruzados, el apareamiento de fecha y hora están escritas en ambos las placas y láminas de la. Las placas se almacenan 5 a 7 días y se puede mantener más tiempo, aunque la eficiencia de la germinación cigoto disminuye progresivamente. </li></ol> Parte 3: Disección Tétrada <ol><li> Las células vegetativas debe ser quitado manchas secas de la mezcla de apareamiento con una cuchilla sin filo, como un bisturí. La hoja puede ser utilizada para exponer un área de cigotos, que se adhieren firmemente al agar al 3%. Este paso se realiza generalmente en la tarde, de modo que los cigotos han germinado a la mañana siguiente, pero no han sido objeto de una segunda división, vegetativa,Rinde 8 progenie. biólogos Chlamydomonas rápidamente aprenden a reconocer los cigotos en el microscopio, ya que son más grandes que las células vegetativas, y cuentan con una pared gruesa zigospora que da un contorno negro. Zygospores a menudo presentan un color amarillo, pero también puede ser un poco verde. El personaje más importante es que se adhieren a agar, mientras que las células vegetativas son fácilmente a un lado. Sin embargo, si las placas son demasiado seca no será el caso. Además, si la mezcla de apareamiento era demasiado densa, los cigotos se puede formar una alfombra de lo que hará que la transferencia de cigotos persona difícil o imposible. Densidades apropiadas se establecen en el paso 2.1, y un rápido aprendizaje de la experiencia. </li><li> Agujas de vidrio se preparan para la recolección y separación de los cigotos progenie tétrada. Estos son preparados por tirar 3 varillas de vidrio mm en una pequeña llama para generar un hilo largo y delgado, que puede ser roto. Las puntas de estos extremos afilados o se suavizan doblado en los ganchos, por el calentamiento breve a fuego lento (por ejemplo, alcohol o una cerilla encendida). Prepare 3 o más agujas de vidrio, ya que se rompen con bastante facilidad, sobre todo para usuarios inexpertos. </li><li> Cigotos se reunieron en el 3% de TAP-Difco placa con una aguja de cristal en un pequeño montón. Un cuadrado de agar (hasta 0,5 cm 2) que contiene los cigotos se forma y se extirpa con un escalpelo aburrido. A continuación, el bloque de agar se coloca boca abajo en un 15 mm 1,5% TAP-Difco placa, y se deslizó a lo largo de una línea horizontal de 1,5 cm desde la parte superior de la placa, la distribución de los cigotos. La línea horizontal superior, y una red con aproximadamente 1,5 cm horizontal y 0,5 cm de separación verticales son grabadas en la parte posterior de las placas. La red será utilizada al día siguiente como una guía para la separación de la progenie tétrada (la red también puede ser grabado al agua fuerte el día de la disección). Finalmente, los cigotos individuales se mueven a las intersecciones de las líneas horizontal superior, con cada línea vertical, con unos 20-30 cigotos por placa. Aunque sólo el 12 o así será disecado, no todos los cigoto germina, y algunos se divide dos veces antes de estallar, por lo cigotos adicionales son necesarios. </li><li> Después de la distribución de los cigotos, y las células vegetativas que se han transferido de forma accidental la placa de agar 3% son asesinados por la exposición de la placa invertida durante 30 segundos sobre un plato de cristal que contienen una capa delgada (1.2 mm) de la capa de cloroformo. La distancia entre la placa y el cloroformo deben quedar unos 5 cm. Si el cloroformo se usa demasiado, o si la distancia es demasiado grande o pequeño, se corre el riesgo ya sea matando a los cigotos o no matar a las células vegetativas. El tratamiento de cloroformo se debe hacer inmediatamente después de la zygospores se han distribuido en la placa de la germinación, para garantizar la selectividad. </li><li> La germinación tarda entre 16-20 horas para la mayoría de las cepas, pero puede variar con el genotipo y la edad de los cigotos. Para la germinación, las placas se colocan bajo condiciones de poca luz (239 NE / m 2 / seg), o en la luz atenuada medio cubriendo la placa con un pañuelo. </li><li> Cigotos germinar se pueden identificar con el microscopio las células inflamadas o estallar a veces con células hijas dentro de ellos. Si el zigospora no se ha roto, tocando suavemente con una aguja de vidrio se rompe la pared si se ha producido la germinación. Las células hijas son suavemente arrastrado a los puntos de la red por debajo del cigoto. Es más fácil hacer esto mediante la captura de la célula en una pequeña cantidad de medio líquido liberado del agar por la presión de la aguja de vidrio, y arrastrando el &quot;charco&quot; detrás de la aguja. Una forma de &quot;seguro&quot; de que uno ha diseccionado un cigoto y no una célula vegetativa que se divide en dos ocasiones, es que aparte de las células hija de cuatro, la &quot;piel&quot; de la pared zigospora seguirá siendo. </li><li> La progenie disecados se cultivan en condiciones adecuadas hasta clonies visibles pueden ser recogidos para su posterior análisis. Las placas deben ser controlados diariamente por la contaminación. </li></ol> Nota: La generación de diploides vegetativo no se describe aquí, pero básicamente consiste en la mezcla de revestimiento de apareamiento en un medio que no va a apoyar el crecimiento de cualquier cepa parental, pero apoyará el crecimiento de la diploide (por ejemplo, dos marcadores auxotropic diferentes). Las placas se mantienen a la luz para evitar la formación de cigotos.

<ol>
	<li>Harris, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chlamydomonas+Sourcebook:+Introduction+to+Chlamydomonas+and+Its+Laboratory+Use.">The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and Its Laboratory Use.</a> Conf Proc IEEE Eng Med Biol Soc. , 119-273 (2005).</li></ol>

The authors have nothing to disclose.

El análisis genético es simple en Chlamydomonas, pero requiere de técnicas específicas para generar gametos y progenie tétrada separado. Con cepas sanas, las técnicas son fáciles de adquirir. Con algunas cepas mutantes, se convierte más en un arte, y los lectores deben consultar el volumen 1 del Libro recientemente publicado Chlamydomonas de notas históricas y consejos prácticos. Además, debe tenerse en cuenta que hay muchas variaciones sobre el tema básico descrito anteriormente, ...

<!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>CC-125</td>
<td>Mating type plus strain</td>
<td>Chlamydomonas Stock Center</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CC-124</td>
<td>Mating type minus strain</td>
<td>Chlamydomonas Stock Center</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tris-Acetate-Phosphate (TAP) </td>
<td>Growth medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>N10</td>
<td>Growth medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Difco agar</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fisherbrand Stainless-Steel Blades</td>
<td>Tools</td>
<td>Fisher Scientific</td>
<td>08-916-5B</td>
<td>&nbsp;</td>
<tr>
<td>BD Surgical Blade Handles</td>
<td>Tools</td>
<td>Fisher Scientific</td>
<td>08-914-5</td>
<td>&nbsp;</td>
<tr>
<td>Fisherbrand Metal Scalpels</td>
<td>Tools</td>
<td>Fisher Scientific</td>
<td>08-920A</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

mating and tetrad separation of chlamydomonas reinhardtii for genetic analysis

El alga verde unicelular<em&gt; Chlamydomonas reinhardtii</em&gt; (<em&gt; Chlamydomonas</em&gt;) Se ha convertido en un organismo popular para la investigación en diversas áreas de biología celular y genética, debido a su ciclo de vida simple, facilidad de crecimiento y la manipulación para el análisis genético, recursos genómicos, y capacidad de transformación del núcleo y orgánulos ambos. Cepas de apareamiento es una práctica común cuando los métodos genéticos se utilizan en<em&gt; Chlamydomonass</em&gt;, Para crear diploides vegetal para el análisis de la dominación, o después de la disección tétrada a la herencia determinar nuclear vs organellar, para poner a prueba alelismo, para analizar epistasy, o para generar poblaciones con el objeto de mapa basado en la clonación. Además, los cruces genéticos se utilizan habitualmente para combinar genotipos organellar con especial genotipos nuclear. Aquí nos demuestran los métodos estándar para la gametogénesis, el apareamiento, la germinación de cigoto y la separación tétrada. Este protocolo consiste en una serie fácil de seguir los pasos que harán que los enfoques genéticos susceptibles de científicos que están menos familiarizados con<em&gt; Chlamydomonas</em&gt;. Parámetros clave y los puntos conflictivos se explican. Finalmente, los recursos para obtener más información y los métodos alternativos se proporcionan.

Watch this Scientific Journal Video about Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis at JoVE.com

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Apareamiento y la separación tétrada se requieren para el análisis genético en<em&gt; Chlamydomonas reinhardtii</em&gt;. Aquí nos demuestran los métodos estándar para la gametogénesis, el apareamiento, la germinación de cigoto y la disección tétrada. Este protocolo consiste en una serie fácil de seguir los pasos que harán que los enfoques genéticos susceptibles de científicos que están menos familiarizados con<em&gt; Chlamydomonas</em&gt;.

El apareamiento y la separación de la Tétrada Chlamydomonas reinhardtii Para el análisis genético

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

mating-tetrad-separation-chlamydomonas-reinhardtii-for-genetic

Research

JoVE Journal

Biology

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

19.8K vistas.  Cornell University. Apareamiento y la separación tétrada se requieren para el análisis genético en Chlamydomonas reinhardtii. Aquí nos demuestran los métodos estándar para la gametogénesis, el apareamiento, la germinación de cigoto y la disección tétrada. Este protocolo consiste en una serie fácil de seguir los pasos que harán que los enfoques genéticos susceptibles de científicos que están menos familiarizados con Chlamydomonas.

Video: Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Luz / oscuridad de transición de prueba para ratones

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Investigación

Biología

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para la Cultura de primeros embriones del polluelo ex vivo (Nueva Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Monte doble entero hibridación in situ de embriones de pollo temprano

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Método para la tinción de anticuerpos enteros en el monte del polluelo

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Ensayo de inducción neural en el embrión de pollo

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 ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

La disección de Gyrus dentado del hipocampo de ratones adultos

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

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 ...

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 ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

ADN electroporación, aislamiento e Imagen de miofibras

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...