Este estudio fue apoyado por el NIH R00HL102154, y la Sociedad Americana de Hematología premio académico a P Ji.

Los experimentos descritos en este protocolo se realizaron de acuerdo con las directrices y normas establecidas por la Universidad Northwestern Institucional Cuidado de Animales y el empleo Comisión. 1 Preparación del Medio de Cultivo <ol><li> Prepare la solución de fibronectina. Añadir 1 ml de agua a un vial de fibronectina humana (1 mg). Deje la solución en la campana de cultivo de tejido durante 30 minutos sin agitación. Transferir el líquido total a 50 ml de PBS para hacer una concentración final de 20 mg / ml y mezclar suavemente también. Hacer la solución de fibronectina fresco antes de recubrir la placa (ver más abajo). </li><li> Preparar 50 ml de medio libre de Epo (medio SCF). Combinar los ingredientes enumerados en la Tabla 1. El medio se puede almacenar a 4 ° C durante 1 mes cuando recién hecho. </li><li> Preparar 50 ml de medio que contiene Epo. Combinar los ingredientes enumerados en la Tabla 2. El medio se puede almacenar a 4 ° C durante 1 mes cuando recién hecho. </li></ol&gt; 2. fibronectina placa recubierta <ol><li> Añadir 1 ml de solución de fibronectina a cada pocillo de una placa de 6 pocillos (o 0,5 ml a cada pocillo de una placa de 12 pocillos). Deja la placa en la campana de cultivo de tejido durante 1 hora. </li><li> Enjuague pozos con agua estéril dos veces y secar la placa. </li><li> Envuelva la placa y guardar en la cámara frigorífica a 4 ° C. </li></ol> 3. Purificación del Hígado Fetal Eritroblastos NOTA: Use el hígado fetal de E13 a E15 cronometrados ratones preñados (C57BL / 6). Se prefieren los hígados E13.5 para maximizar el rendimiento de CFU-E progenitores. Para obtener los ratones preñados cronometrados, de 6 a 8 semanas de edad ratones machos y hembras fueron colocados en una jaula (generalmente un macho con una o dos hembras). Se verificaron tapones vaginales hembra a la mañana siguiente para determinar si el apareamiento tuvo éxito. El primer día de la gestación fue el día después de que se encontró el enchufe. <ol><li> Preparación de las células totales de hígado fetal <ol><li> Sacrificar el mouse por inhalación de CO2 y dislocación cervical. Pulverizar el abdomen con etanol al 70% para la desinfección. Abra el abdomen con unas tijeras de disección para extraer el útero. Lave el útero en 1x PBS. </li><li> Diseccionar los fetos en condiciones estériles utilizando una campana de cultivo de tejidos y herramientas tratados en autoclave y lavar en PBS 1x. </li><li> Diseccionar los hígados fetales de los fetos. Sostenga el cuerpo del feto con una pinzas, tire suavemente el hígado fetal (de color rosa) lejos del cuerpo con otros fórceps. Limpiar el hígado fetal con las pinzas para quitar los tejidos fibróticos asociados. Coloque todos los hígados fetales en 1x PBS fresco que contenía suero bovino fetal al 10%. </li><li> Mecánicamente interrumpir hígados fetales pipeteando arriba y abajo. </li><li> Se filtra la suspensión celular a través de un filtro de células de 40 m en un tubo cónico de 50 ml. Sedimentar las células a 800 xg durante 5 min. </li><li> Aspirar el sobrenadante y resuspender las células en 1 ml de PBS con 10% de FBS (aproximadamente 8 E13.5 hígados fetalespor ml). </li></ol></li><li> Purificación de Ter119 Eritroblastos negativos <ol><li> Bloquear las células con IgG de rata (0,2 mg / ml) en hielo durante 15 min. </li><li> Sin lavado o centrifugación, añadir anticuerpos anti-TER119 biotinilado a la suspensión celular (1 g / ml). Incubar durante 15 min en hielo. </li><li> Lavar en 1x PBS con 10% de FBS (01:10) y se centrifuga a 800 xg durante 5 min. Resuspender las células en 1 ml de PBS con 10% de FBS. </li><li> Añadir 75 l estreptavidina conjugada con partículas magnéticas y se incuba durante 10 minutos en hielo. </li><li> Añadir el volumen a 2,5 ml con PBS que contenía 10% de FBS. Transferir la suspensión celular en un polipropileno de 5 ml ronda tubo inferior. </li><li> Inserte el tubo en un aparato de clasificación de células magnéticas y se incuba durante 10 min. Ter119 células positivas se unirán al lado del tubo. Los eritroblastos negativos Ter119 unattached permanecerán en la suspensión. </li><li> Verter la suspensión de células en un nuevo polipropileno de 5 ml ronda tubo inferior. </li><li><html> Repetir los pasos 3.2.6 y 3.2.7 para eliminar las células positivas Ter119 residual. </li><li> Sedimentar las células a 800 xg durante 5 min. Aspirar el sobrenadante. Resuspender las células en 1 ml de PBS que contenía 10% de FBS y contar células vivas con azul de tripano. </li></ol></li></ol> 4. transducción con virus cDNA de codificación o shRNA <ol><li> Placa de las células hepáticas fetales negativos Ter119 purificada a 3-5 x 10 5 / pocillo en una fibronectina recubierto placa de 12 pocillos (ajustar el número de células si se utiliza una placa diferente). </li><li> Añadir polibreno a una concentración final de 10 mg / ml para facilitar la transducción viral. Girar infectar las células con lentivirus o retrovirus, ADNc codificación o shRNA, a 800 xg durante 1-1,5 horas a 37 ° C. </li></ol> 5. Cultura de los transducidas Ter119 mouse Negativo fetales Eritroblastos Hígado <ol><li> Para probar las funciones de genes en la etapa temprana de la eritropoyesis terminal (Figura 1 líneas discontinuas).&quot;&gt; </ol.><ol><li><ol. li=""> Cultivar las células transducidas en medio libre de Epo (medio SCF) durante 12 horas para permitir la expresión de los genes y el mantenimiento del estado progenitor transducidas. </ol.></li><li> Centrifugar las células a 800 xg durante 5 min. Aspirar el sobrenadante. Resuspender las células en 1 ml de Epo que contenían medio en cada pocillo de una placa de 12 pocillos. </li><li> Después del cultivo durante 24 o 48 horas, recoger las células para ensayos adicionales. </li></ol></li><li> Funciones de los genes de prueba en la etapa tardía de la eritropoyesis terminal (Figura 1 líneas continuas). <ol><li> Cultivar las células transducidas inmediatamente en Epo contiene medio. </li><li> Después del cultivo durante 24 o 48 horas, recoger las células de los ensayos adicionales. </li></ol></li></ol> 6. La tinción de las células cultivadas de hígado fetal para el análisis por citometría de flujo <ol><li> Lavar y resuspender las células en 1x PBS. Cosecha 2 x 10 5 células cultivadas para el análisis de citometría de flujo. </li><li> Incubarlas células con anticuerpos fluoróforo conjugado durante 20 min a temperatura ambiente en la oscuridad. Para probar la diferenciación, utilizar APC conjugado anti-CD71 PE y anti-TER119 conjugado. Para probar la enucleación, usar Hoechst 33342 tinción además de los otros anticuerpos. </li><li> Lavar las células con PBS 1x. Resuspender las células en 0,5 ml de PBS que contenía 1% de FBS y yoduro de propidio (PI) (1 mg / ml) para teñir las células muertas y excluirlos del análisis. </li><li> Flujo de Conducta análisis cytrometric. Analice las células de control individuales de color teñido y sin teñir primero para el ajuste y compensación del citómetro de flujo. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Aquí presentamos un sistema único para analizar cronológicamente ratón fetal eritropoyesis terminal de hígado. A través de la aplicación de diferentes condiciones de cultivo, disecado éxito eritropoyesis terminal en fases tempranas y tardías. Esto es particularmente importante para determinar los mecanismos de genes con múltiples funciones. Por ejemplo, GTPasas Rac juegan papeles importantes en diferentes etapas de la eritropoyesis terminal. La inhibición de Rac GTPasas en la etapa temprana de la diferenciaci...

La eritropoyesis es el proceso de diferenciación de células madre hematopoyéticas multipotentes a eritrocitos maduros. Este proceso por etapas incluye la formación de unidades de ráfaga eritroides comprometidos de formación (BFU-ES), las unidades que se dividen rápidamente eritroides formadoras de colonias (CFU-ES), y morfológicamente reconocibles eritroblastos 1,2. Eritropoyesis Terminal de células progenitoras CFU-E implica eritropoyetina dependiente secuencial etapas independientes y 2,3. En la etapa temprana de la eritropoyesis terminal, eritropoyetina (EPO) se une a su receptor en la superficie celular e induce una serie de vías de señalización corriente abajo que impiden la apoptosis celular y promueven divisiones celulares rápidas y expresión génica 1,4. En la etapa tardía de la eritropoyesis terminal, eritroblastos se someten a la salida del ciclo celular terminal, y la condensación de la cromatina núcleo, y la extrusión de los núcleos altamente condensados ​​5. Nuestra comprensión de la terminaleritropoyesis ha mejorado mucho en las últimas décadas, que es en gran parte debido a la utilización con éxito de varios in vitro e in vivo en modelos de ratones de 6-9. Entre estos modelos, el cultivo in vitro de ratón fetal eritroblastos hígado proporciona muchas ventajas, incluyendo la facilidad de purificación celular, proliferación y diferenciación rápida, y un cerrador imitan a la eritropoyesis humana 10,11. En este sistema, un gran número de células progenitoras eritroides a partir de hígados fetales de ratón pueden ser fácilmente aislados por la purificación solo paso de TER119 (un marcador para las células eritroides maduras) eritroblastos negativos. Durante el cultivo de dos días de los eritroblastos, la diferenciación de estas células puede ser monitoreado por un análisis de citometría de flujo basado en la expresión de superficie de receptor de transferrina (CD71) y el antígeno de TER119 12. Además, la enucleación de los eritroblastos de diferenciación terminal puede ser detectada por un fabricante de ADN (Hoechst 33342) 13. Además, los progenitores purificados se pueden modificar genéticamente por la expresión exógena de ADNc o pequeños ARN de horquilla (shRNAs) para los genes de interés, lo que facilita los estudios mecanísticos de las funciones de la expresión génica en la eritropoyesis 11,13,14. Por otra parte, la tasa de crecimiento celular rápido puede ser una espada de doble filo, ya que es difícil de caracterizar las funciones de genes en diferentes etapas de la eritropoyesis terminal. En la mayoría de los casos, es difícil determinar si un funciones de genes específicos en la etapa temprana de la eritropoyesis desde el terminal en el momento en el ADNc o shRNAs expresaron, las células ya han pasado la etapa temprana. Para solucionar este problema, hemos desarrollado un sistema único para diseccionar las etapas tempranas y tardías de la eritropoyesis terminal. Para la etapa temprana de la eritropoyesis terminal, modificados genéticamente Ter119 eritroblastos negativos fueron cultivadas en medio libre de Epo-pero que contiene el factor de células madre (SCF), IL-6 y FLT3ligando para mantener su estado progenitor y deje los ADNc transducidas o shRNA de expresión 13. Las células se cambiaron a medio que contiene Epo después de 12 horas para inducir la proliferación y diferenciación celular. De esta manera, cuando las células empezaron a diferenciar, los ADNc fueron transducidas o shRNAs ya expresados. Para la etapa tardía de la eritropoyesis terminal, Ter119 eritroblastos negativos Epo fueron cultivadas en medio que contiene inmediatamente después de la transducción. Por lo tanto, se puede analizar las funciones de los genes de interés en la etapa tardía de la eritropoyesis terminal. En resumen, una amplia aplicación de este sistema ayudaría a las funciones de genes Dissect en diferentes etapas de la eritropoyesis terminal.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s MediumIMDM (IMDM)</td>
			<td>Gibco</td>
			<td>12440-053</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GEMINI Bio-product</td>
			<td>700-102P</td>
			<td></td>
		</tr>
		<tr>
			<td>b-mecaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td>0.1 M in IMDM,&#160; 4&#8451;stock</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>100 X</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Hyclone</td>
			<td>SH30034.01</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>Stem cell factor (SCF)</td>
			<td>STEMCELL TECH</td>
			<td>2630</td>
			<td>100 ng/ul, -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Mouse FIT3 Ligand (Flt-3L)</td>
			<td>BD Biosciences&#160;</td>
			<td>14-8001-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Recombinant Interleukin-6 (IL-6)&#160;</td>
			<td>GEMINI Bio-product</td>
			<td>300-327P</td>
			<td></td>
		</tr>
		<tr>
			<td>fibronectin</td>
			<td>BD Biosciences&#160;</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>STEMCELL TECH</td>
			<td>9300</td>
			<td>10% stock in IMDM</td>
		</tr>
		<tr>
			<td>Insulin solution, human</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td>4&#8451; stock</td>
		</tr>
		<tr>
			<td>holo-transferrin human</td>
			<td>Sigma</td>
			<td>T0665</td>
			<td>50 mg/ml in Water -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Erythropoietin(Epo)</td>
			<td>PROCRIT</td>
			<td>NOC 59676-303-00</td>
			<td>3,000 U/ml stock</td>
		</tr>
		<tr>
			<td>Streptavidin particles Plus</td>
			<td>BD Pharmingen</td>
			<td>557812</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>STEMCELL TECH</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Round-Bottom Tube, 5 ml</td>
			<td>FALCON</td>
			<td>352063</td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis

La eritropoyesis implica un proceso dinámico que comienza con unidades de ráfaga eritroides comprometidos formación (BFU-Es), seguido de rápida división de colonias eritroides unidades (UFC-E). Después de CFU-E, las células son morfológicamente reconocibles y generalmente denominado eritroblastos terminales. Uno de los retos para el estudio de la eritropoyesis de terminal es la falta de enfoques experimentales para diseccionar las funciones de genes de manera cronológica. En este protocolo, se describe una estrategia única para determinar las funciones de genes en las etapas tempranas y tardías de la eritropoyesis terminal. En este sistema, el ratón TER119 hígado fetal (marcador de células eritroides maduro) eritroblastos negativos fueron purificados y transducidas con la expresión exógena de los ADNc o pequeños ARN de horquilla (shRNAs) para los genes de interés. Las células se cultivaron posteriormente en medio que contiene factores de crecimiento distintos de eritropoyetina (Epo) para mantener su etapa progenitor durante 12 horas al tiempo que permite los ADNc exógenos o shRNAsexpresar. Las células se cambiaron a medio Epo después de 12 horas para inducir la diferenciación celular y la proliferación, mientras que los materiales genéticos exógenos ya se expresaron. Este protocolo facilita el análisis de las funciones de genes en la etapa temprana de la eritropoyesis terminal. Para estudiar finales de la eritropoyesis terminal de etapa, las células se cultivaron inmediatamente en un medio de Epo después de la transducción. De esta manera, las células ya se diferenciaron a la etapa tardía de la eritropoyesis terminal cuando se expresaron los materiales genéticos transducidas. Se recomienda una aplicación general de esta estrategia que ayudaría a entender las funciones de genes detalladas en las diferentes etapas de la eritropoyesis terminal.

La figura 1 resume las estrategias experimentales. El protocolo consiste en dos condiciones independientes para dirigir las funciones de las moléculas de señalización en las etapas tempranas y tardías de la eritropoyesis terminal. Ter119 eritroblastos fetales de hígado negativos fueron purificados de feto de ratón E13.5. Análisis de citometría de flujo de células eritroides fetales del hígado antes y después de la purificación demostraron que la purificación era eficiente (Figuras 2...

Watch this Scientific Journal Video about Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis at JoVE.com

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Presentamos un ratón sistema de cultivo in vitro fetal eritroblasto hígado que disecciona las etapas tempranas y tardías de la eritropoyesis terminal. Este sistema facilita el análisis funcional de genes específicos en diferentes etapas de desarrollo.

Ratón Sistema Cultura Hígado Fetal de diseccionar las funciones de genes objetivo en las primeras y últimas etapas de la Terminal de la eritropoyesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

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Research

Education

JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

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14.8K vistas.  Northwestern University.  Presentamos un ratón sistema de cultivo in vitro fetal eritroblasto hígado que disecciona las etapas tempranas y tardías de la eritropoyesis terminal. Este sistema facilita el análisis funcional de genes específicos en diferentes etapas de desarrollo. 

Video: Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Investigación

Inmunología e Infección

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Uso del ratón humanizado BLT como un modelo de células madre del tumor Terapia génica basada en

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

Multi-objetivo enfoque de procesamiento paralelo para Gene a la estructura de determinación de la polimerasa subunidad PB2 Influenza

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Vaccinia Virus Reporter para cuantificar la función viral en todas las etapas de la Expresión Génica

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Aislamiento de leucocitos a partir de los tejidos murinos en la interfase materno-fetal

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

Los cultivos humanos de la placenta y Decidual de órganos para el Estudio de Infecciones en la interfase materno-fetal

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Ensayo de citometría de flujo de base para la supervisión de funciones de las células NK

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Desarrollo de un sistema reportero virus de la hepatitis B para supervisar las primeras etapas del ciclo de replicación

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Generación de variantes de Escape de neutralizar anticuerpos Monoclonal del Virus de la Influenza

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Un método para evaluar las funciones efectoras mediadas por Fc inducidas por los anticuerpos específicos de la hemaglutinina de la gripe

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...