Este trabajo fue apoyado por las becas de investigación de la Facultad de la Facultad de Artes y Ciencias Dean oficina en la Universidad de Hartford.

Durante el estudio, tratamiento y cuidado de los animales el cumplimiento de la política de los institutos nacionales de salud, estaban de acuerdo con las directrices y fueron aprobados por el Comité de uso y cuidado Animal institucional Animal de la Universidad de Hartford.
1. arrastre de animales
Nota: Se utilizan ratones J B6129SF2 machos de veinte semanas de edad en el estudio.
<ol><li>Arrastrar a ratones a una 12 h de luz (luz estándar blanca arriba) / 12 h oscuro ciclo durante 2 semanas antes del experimento. Nota: Aquí tiempo zeitgeber (ZT) 0 corresponde a luces y ZT12 a luces, manteniendo otros factores ambientales (es decir, alimentos, agua y temperatura) constante 21.</li>
</ol>2. preparación de instrumentos, medio de cultivo y medio de desafío
<ol><li>Autoclave con fórceps y tijeras de disección. Abrigo de pares de portaobjetos esmerilado en papel de aluminio y autoclave.</li>
	<li>Preparar medio de cultivo mediante la adición de suero bovino fetal (FBS) de RPMI 1640 a una concentración final de 10%. Preparar 10 mL de medio de desafío en tubos de 50 mL mediante la adición de los siguientes pmap al medio de cultivo (RPMI con 10% FBS); LPS (5 μg/mL), ODN1826 (5 μg/mL), calor-matado Listeria monocytogenes (108 HKLM/mL), u otro PAMP en su concentración sugerida.</li>
	<li>Calentar el medio de cultivo y el medio reto a 37 ° C en un baño de agua y mantener aproximadamente 70 mL de tampón de fosfato estéril salino (PBS, pH 7,2) a temperatura ambiente.</li>
	<li>Añadir 10 mL de medio de cultivo utilizando una pipeta de 10 mL y ayuda de la pipeta a un tubo de 50 mL y coloque en hielo.</li>
	<li>Agregar aproximadamente 30 mL de etanol al 70% a un 100 mL vaso de precipitados y lugar extremos de tijeras y pinzas de disección en el vaso para evitar la contaminación microbiana.</li>
	<li>Preparación tampón de lisis para aislamiento de RNA mediante la adición de 10 μl β-mercaptoetanol (bajo una campana de humos) cada 1 mL de tampón de RLT en un tubo de 50 mL. Hacer sólo la cantidad que sea necesaria (600 μl por muestra, que consta de aproximadamente 1 x 106 células). Nota: El RLT amortiguador es un componente del kit de extracción de RNA que apoya la Unión de la RNA a la membrana de silicona.</li>
</ol>3. Splenocyte aislamiento y desafío
<ol><li>Eutanasia a ratones en un momento particular zeitgeber vía narcosis por mantener en su caja original y adición de CO2 a la jaula con un caudal de 3 L/min continuar suministro de CO2 durante 1 minuto después de la respiración se detiene.</li>
	<li>Confirman muerte por dislocación cervical, colocando el pulgar y el dedo índice a ambos lados del cuello en la base del cráneo. Alternativamente, presione una varilla en la base del cráneo empujando rápidamente (con la otra mano) la base de la cola o las extremidades posteriores para causar la separación de las vértebras cervicales del cráneo22.</li>
	<li>Rociar el tronco de ratón con etanol al 70% y limpie con una toalla de papel. Coloque el ratón sobre su espalda y ligeramente inclinada hacia su lado derecho. Cortar la piel, utilizando disección tijeras, a lo largo del lado izquierdo del ratón, sobre a medio camino entre la parte delantera y patas traseras.</li>
	<li>Con unas pinzas, agarra el peritoneo y cuidadosamente hacer una incisión para no dañar el bazo. Extirpar el bazo con pinzas y coloque en un tubo estéril de 50 mL que contenga aproximadamente 10 mL de medio de cultivo en el hielo. Repita para el resto de los animales. Nota: El bazo es el color de un frijol, y es más largo y más planas que el riñón.</li>
	<li>
Transferencia de un bazo con 2 mL de medio de cultivo para un pequeño plato de Petri estéril.
	<ol>
<li>Homogeneizar el bazo por molienda entre la porción helada de dos portaobjetos esmeriladas estériles. Si es posible, mantener el tema y las células en el medio durante el proceso de homogeneización.</li>
		<li>Una vez bien homogeneizado, pipeta de 2 mL de medio de cultivo con los esplenocitos a través de un tamiz de célula 40 μm nylon en un tubo de 50 mL.</li>
		<li>Repita los pasos anteriores para los bazos restantes usando nuevos pares de diapositivas helados, tubos de 50 mL con medio de cultivo y los filtros de la célula.</li>
	</ol>
</li>
	<li>Añada 8 mL de medio de cultivo frío a cada uno de los tubos de 50 mL que contiene los esplenocitos para un volumen total de 10 mL/tubo. Determinar el número de células por mililitro usando un hemocitómetro.</li>
	<li>Añadir aproximadamente 1 x 106 células/pozo en placas de cultivo de 6 pozos. Añadir las células a la cantidad de pozos que corresponden al número de diferentes PAMPs para el experimento e incluyen un control también. Añadir 3 mL de medio de cultivo o 3 mL de medio de desafío a los pocillos respectivos.</li>
	<li>Incubar las placas a 37 ° C en 5% CO2 para 3 h.</li>
	<li>Raspar las células de la parte inferior de la bien con una punta de pipeta 1.000 μl conectado a una micropipeta P1000. Transfiera el medio que contiene las células con el mismo 1.000 μl Punta de la pipeta en un tubo de 15 mL.</li>
	<li>Sedimenten las células mediante centrifugación a 167 x g durante 5 min a temperatura ambiente. Quite el sobrenadante y lavar el sedimento celular con 5 mL de PBS.</li>
	<li>Sedimenten las células una segunda vez a 167 x g durante 5 minutos, retirar el sobrenadante y añadir 600 μl de tampón de lisis para el precipitado de células con el fin de lisar las células. Luego proceder al aislamiento de RNA.</li>
</ol>4. aislamiento RNA y síntesis del cDNA
<ol><li>Aislar RNA de esplenocitos utilizando el kit de extracción de RNA según instrucciones del fabricante y realizar la digestión de ADN en la columna 'opcional'.</li>
	<li>Antes de la síntesis de cDNA, determinar la concentración de RNA utilizando un espectrofotómetro microvolume para verificar que la concentración esté dentro del rango óptimo de RNA (2 μg) del kit de síntesis de cDNA.</li>
	<li>
Sintetizar cDNA para cada una de las muestras mediante el kit de transcripción inversa según las instrucciones del fabricante. Utilice 10 μl de RNA para cada una de las muestras en un volumen total de reacción de 20 μl. En la terminación de la transcripción inversa (termociclador ejecutar) añada 20 μL H2O para cada reacción.
</li>
	<li>Construir la curva estándar utilizando una micropipeta P10 o P20 a mRNA de unas muestras de control (por ejemplo, 5 μl de 2 muestras para un total de 10 μl) en un tubo de 0,5 mL para preparar el cDNA.<ol>
<li>Añadir 10 μl de 2 x mix master la transcriptasa reversa.</li>
		<li>En la terminación de la transcripción inversa, añadir 10 μl de H2O para el tubo de reacción, que servirá como la concentración inicial ("1") en la serie de diluciones de la curva estándar.</li>
		<li>Realizar una serie de diluciones 10 veces (1 a 10-4) mediante la adición de 45 μl de agua utilizando una micropipeta P100 o P200 en cuatro tubos de 0.5 mL señalados 10-1, 10-2, 10-3y 10-4.<ol>
<li>Añadir 5 μl de la concentración inicial "1" en el primer tubo (10-1) utilizando una micropipeta de P20, mezclar mediante pipeteo arriba y abajo varias veces, luego transferir 5 μl del tubo de 10-1 en el tubo señalado 10-2 y mezclar.</li>
			<li>Transferencia de 5 μl utilizando una micropipeta P20 del tubo 10-2 en el tubo señalado 10-3 y mezclar. Transferencia de 5 μl utilizando una micropipeta P20 del tubo 10-3 en el tubo señalado 10-4 y mezclar.</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. cuantitativo reacción en cadena de polimerasa (qPCR)
<ol><li>Determinar la cuantificación relativa de niveles de mRNA por qPCR. Dentro de la instalación experimental, seleccione "Cuantificación – curva estándar relativa" y "TaqMan" química. Cambio el volumen de reacción de 10 μl. entrar la información relevante en el diseño de la placa (por ejemplo, gene de la blanco, curva estándar, reportero, etc.).</li>
	<li>Preparar la reacción para que contenga 0,5 μl del ensayo primer punta de prueba, 5 μl de la mezcla principal de expresión génica, 2 μl de H2O y 2.5 μl de cDNA (10-100 ng).<ol>
<li>Preparar una mezcla maestro multiplicando cada uno de los reactivos anteriores (excepto el cDNA) por el número de reacciones, asegurándose de incluirlos de la curva estándar, controles negativos, actuando cada reacción en duplicado y 2 reacciones extras que será para la variación del pipeteo.</li>
		<li>Pipeta 7.5 μl de la mezcla principal dispuesta en pozos determinados en una placa de 96 pocillos de reacción con una micropipeta o una pipeta multicanal. Pipeta de 2.5 μl de cDNA en el bien apropiado para las incógnitas y las normas y 2.5 μl de H2O en los pocillos de control negativo.</li>
		<li>Incluyen ensayos de primer punta de prueba de varios genes de reloj molecular y también incluyen un ensayo para un control endógeno (p. ej., β-actina).</li>
	</ol>
</li>
	<li>Con el fin de determinar los valores de expresión relativa, calcular la cantidad relativa media para cada repetición, después, para cada muestra, divida la cantidad relativa media para el gene de la blanco por la cantidad relativa promedio de β-actina.</li>
</ol>6. estadístico análisis
<ol><li>Usando el software de análisis estadístico, introducir datos en el programa en la opción estadísticas de columna. Seleccione un unidireccional ANOVA con prueba post-hoc de Dunnett para determinar diferencias entre los valores medios de la expresión de genes reloj después del desafío PAMP versus el control.</li>
</ol>

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El autor no tiene nada que revelar.

Dentro de este protocolo, puede utilizarse un espectrofotómetro microvolume para cuantificar y evaluar la pureza del ARN se utiliza en la determinación de la expresión génica. Los ácidos nucleicos absorben UV luz a 260 nm, típicamente las proteínas absorben la luz a 280 nm, mientras que otros potenciales contaminantes durante un procedimiento de extracción de RNA (por ejemplo, fenol) pueden detectarse en 230 nm. Por lo tanto, determinando la absorbancia (A) relación a 260/280 nm (RNA a proteína) y 260/...

El reloj principal en los mamíferos, que organiza las oscilaciones de 24 h para casi todos los aspectos de la fisiología y comportamiento, se encuentra en el núcleo supraquiasmático (SCN) del hipotálamo1,2. Además de regular los procesos biológicos a nivel organismal, el reloj principal también sincroniza los relojes celulares periféricas en todo el cuerpo3,4,5. Mientras que la maquinaria del reloj molecular consiste en por lo menos tres que se enclavija retroalimentación transcripcional traslacional, el núcleo está compuesto por el período (Per1-3), Cryptochrome (Cry1-2), Bmal1, y reloj genes6,7. Además de mantener la sincronización exacta del reloj molecular del núcleo, algunos productos del gene de reloj auxiliares (p. ej., Rev-erbα y Dbp) también regulan la expresión de genes reloj no, es decir, reloj controlado genes (CCGs)6 , 7.
Relojes moleculares funcionales se han descrito en varios tejidos inmunes (p. ej., bazo y ganglios linfáticos)8 y las células (célulasp. ej., B, células dendríticas, macrófagos)8,9. Estas células detectan y responden a patrones moleculares a asociado a patógenos (PAMPs), componentes microbianos conservados, mediante receptores de reconocimiento inmune innato como Toll-like receptores (TLRs)10. Hasta la fecha, se han descrito 13 TLR funcional, que reconocen componentes microbianos como componentes de la pared celular bacteriana, proteína flagelar y los ácidos nucleicos microbianos10. PAMP, lipopolisacárido (LPS), un componente de la pared celular de bacterias Gram-negativas reconocidas por TLR4, se ha demostrado para alterar los ritmos circadianos a nivel organismal y molecular. Por ejemplo, reto en vivo de LPS indujo retrasos de fase luminosa como medida por actividad en ratones11 y condujo a la expresión de gene de reloj reducida en el SCN y el hígado por hibridación en situ y PCR cuantitativa, respectivamente, en las ratas12. Después de un en vivo con LPS, análisis de sangre periférica leucocitos13 y14 de tejido adiposo subcutáneo reveló alterada expresión de varios genes de reloj medido mediante qPCR. Por último, ex vivo desafíos LPS de macrófagos humanos y macrófagos peritoneales de ratón, también alteración reloj expresión medida por qPCR14.
Aquí, describimos un protocolo para evaluar la influencia de los PAMPs LPS, ODN1826 (oligonucleótidos sintéticos que contienen unmethylated CpG motivos) y muerto de calor Listeria monocytogenes (HKLM), reconocido por TLR4 y TLR9 TLR2, respectivamente, en expresión del gene de reloj molecular en esplenocitos de ratón. El protocolo incluye esplenectomía de ratón, splenocyte aislamiento y desafío, RNA extracción, síntesis de cDNA y qPCR para evaluar la expresión de varios genes de reloj. Este protocolo permite la adquisición oportuna de un gran número de células inmunes con muy poco animal o manipulación celular, que puede entonces ser cuestionada ex vivo con pmap varios. El reloj molecular se ha demostrado que modulan los distintos aspectos de la respuesta inmune8,15,16, por lo tanto, la interrupción del reloj molecular probablemente afectaría la correcta variación dependiente del tiempo de la respuesta inmune. Además, desde alteraciones de los ritmos circadianos pueden conducir a patologías graves17,18,19,20, puede ser de interés para los investigadores a cuestionar esplenocitos con una amplia gama de moléculas y valorar su influencia en el reloj.

<table border="0" cellpadding="0" cellspacing="0" style="width:796px;" width="795">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">Frosted slides</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:137px;">12-550-343</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell strainers</td>
			<td>Fisher</td>
			<td align="right">22363547</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipopolysaccharide&#160;</td>
			<td>InvivoGen</td>
			<td>ltrl-eklps</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ODN1826</td>
			<td>InvivoGen</td>
			<td>Tlrl-1826-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HKLM</td>
			<td>InvivoGen</td>
			<td>Tlrl-hklm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Gibco</td>
			<td>20012-043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104 or 74106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-Free DNase Set</td>
			<td>Qiagen</td>
			<td align="right">79254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well cell culture plate</td>
			<td>Denville</td>
			<td>T1006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml tubes</td>
			<td>Corning</td>
			<td align="right">352070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 ml tubes</td>
			<td>Corning</td>
			<td align="right">352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher</td>
			<td align="right">4368814</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays b-actin</td>
			<td>ThermoFisher</td>
			<td>Mm00607939_s1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Per2</td>
			<td>ThermoFisher</td>
			<td>Mm00478113_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Rev-erba</td>
			<td>ThermoFisher</td>
			<td>Mm00520708_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Bmal1</td>
			<td>ThermoFisher</td>
			<td>Mm00500226_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Dbp</td>
			<td>ThermoFisher</td>
			<td>Mm00497539_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR machine StepOnePlus</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>ThermoFisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Fast 96-well reaction plate (0.1 ml)</td>
			<td>ThermoFisher</td>
			<td align="right">4346907</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Statistical Analysis Software</td>
			<td>Prism 7.0a</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression

De comportamiento de la expresión génica, los ritmos circadianos regulan casi todos los aspectos de la fisiología. Aquí, presentamos una metodología para desafiar esplenocitos de ratón con lipopolisacárido (LPS) de patrones moleculares asociados a patógenos (PAMPs), ODN1826 y muertos de calor Listeria monocytogenes y analizar su efecto en el molecular circadiana reloj. Previamente, estudios se han centrado en examinar la influencia de LPS en el reloj molecular usando una variedad de métodos in vivo y ex vivo de una gran variedad de modelos (por ejemplo, ratón, rata y humano). Este protocolo describe el aislamiento y el desafío de esplenocitos, así como la metodología para evaluar reloj gene expresión posterior al desafío mediante PCR cuantitativa. Este enfoque puede utilizarse para evaluar no sólo la influencia de componentes microbianos en el reloj molecular pero también otras moléculas que puede alterar la expresión del reloj. Este enfoque podría utilizarse para embromar aparte el mecanismo molecular de cómo interacción de PAMP-Toll-like receptor influye en la expresión de reloj.

Ratones fueron sacrificados en ZT13, esplenocitos fueron aislados y cuestionada ex vivo con los PAMPs LPS, ODN1826 o HKLM. Después de 3 h, RNA fue aislado y qPCR fue utilizado para evaluar los niveles de expresión relativa de los genes de reloj molecular Clock, Per2, Dbp y Rev-erbα compararon a las células control indiscutido. Después del desafío PAMP, niveles de expresión de reloj no fueron significativamente diferentes de expr...

Watch this Scientific Journal Video about The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression at JoVE.com

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Este protocolo describe una técnica usando esplenocitos de ratón para descubrir patrones moleculares asociados a patógenos que alteran la expresión de genes reloj molecular.

El uso de esplenocitos de ratón para evaluar la influencia de patrón Molecular asociado a patógenos en la expresión de genes reloj

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

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Research

JoVE Journal

Genetics

7.4K vistas.  University of Hartford. Este protocolo describe una técnica usando esplenocitos de ratón para descubrir patrones moleculares asociados a patógenos que alteran la expresión de genes reloj molecular.

Video: The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

La medición paralela de la expresión de genes circadianos del reloj y Hormona en cultivos celulares primarios humanos

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Investigación

Genética

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

Una sola célula de la expresión génica utilizando multiplex RT-qPCR para caracterizar heterogeneidad de las poblaciones linfoides Raras

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

Estudio de la expresión genética regulada por el ciclo celular mediante dos protocolos complementarios de sincronización celular

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

Preparación de muestras y análisis de datos de expresión basada en RNASeq gen de pez cebra

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Una metodología estándar para examinar in situ mutagenicidad en función de la mutación de punto reparación catalizada por CRISPR/Cas9 y SsODN en células humanas

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Producción y purificación de Baculovirus para la aplicación de la terapia génica

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Un método de Optogenetic para controlar y analizar patrones de expresión génica en las interacciones célula a célula

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

Un método para el estudio del polimorfismo C924T del gen Receptor tromboxano A2

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Uso de ovocitos de ratón para evaluar la función humana del Gene durante la Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

Explorar el microbioma de la raíz: extracción de datos de la comunidad bacteriana del suelo, rizosfera y raíz Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...