Este trabajo es apoyado por becas de la Australian National Health and Medical Research Council (NHMRC # 559007) y la Juvenile Diabetes Research Foundation (JDRF 4-2006-1025) y el Plan de Infraestructuras de Operaciones del Gobierno de Victoria. Damos las gracias a los miembros del equipo Tom Mandel Islet Programa de Trasplante de aislamiento de islotes para la prestación de los tejidos humanos. Los tejidos humanos fueron recogidos y utilizados con la aprobación ética local (Hospital de San Vicente HREC-A 011/04 y de la Salud de San Vicente HREC-A 135/08).

1. Preparación de tejido de bazo <ol><li> Nota-todo el material humano debe ser tratado como potencialmente infeccioso y todos los procedimientos deben llevarse a cabo en un gabinete de flujo laminar clase II. El uso de tijeras y pinzas estériles, quitar grasa y tejido fibroso a partir de secciones de bazo (~ 1-2 cm de tamaño) y recortar la mayor cantidad de material de la cápsula exterior como sea posible. </li><li> Cortar un trozo pequeño (1-2 cm 3) de tejido de bazo y colocar cada pieza en un tubo estéril Falcon de 50 ml. </li><li> Snap-congelar los trozos de tejido por inmersión en N 2 líquido. </li><li> Conservar a -80 ° C. Un protocolo similar es adecuado para otros tejidos (s). </li></ol> 2. Preparación de islotes humanos para almacenamiento <ol><li> Islotes Cultura CMRL medios de comunicación. Recoger islotes en un tubo de fondo cónico de 10 ml y lavar dos veces en PBS mediante centrifugación a 1.500 rpm durante 5 min. Retirar el PBS y drenar el buffer residual colocando el tubo invertido brevemente sobre una toalla de papel. Tenga cuidado de nopara desalojar a los islotes. </li><li> Una vez escurrido, vuelva a tapar el tubo y la presión por congelación en nitrógeno líquido y se almacena a -80 ° C. </li></ol> 3. Preparación de Extracto <ol><li> Preparar BAW mix (10:30:60% v / v) y se almacena a 4 ° C. </li><li> Retire el tubo de -80 ° C. Descongelar a temperatura ambiente. </li><li> Añadir suficiente helado BAW para cubrir la pieza de tejido. Para usar islotes 3-5 ml. Para utilizar el tejido de bazo 10-20 ml, dependiendo del tamaño de la pieza de tejido. </li><li> Montar el homogeneizador de tejidos. Limpiar por &#39;homogeneizar&#39; 10-20 ml de 70% etanol / agua. </li><li> Homogeneizar el tejido en múltiples ráfagas, después de colocar la sonda en el tubo homogeneizador con el tejido y la solución BAW. Mantenga el tubo en un cubo de hielo a partir de entonces. </li><li> Limpiar a fondo el homogeneizador entre las muestras, por homogeneización de 10-20 ml de 70% de etanol / agua y luego tampón BAW. Esto evita la contaminación cruzada de tejido entre samples.Dismantleand limpia con 70% de etanol / agua después de nosotrose. </li><li> Si un extracto que contiene sólo el material soluble se requiere, centrifugar extracto de tejido homogeneizado a 4.000 rpm a temperatura ambiente durante 10 min. Si un extracto crudo se requiere más, centrifugar a 1.000 rpm a temperatura ambiente durante 5 min. La técnica más adecuada para la extracción de las proteínas insolubles en BAW depende del análisis de aguas abajo de las proteínas. Para uso en ensayos inmunológicos funcionales que sugeriría intentar disolver la fracción insoluble en urea 8 M como previamente hemos encontrado que esto se tolera bien 6. </li><li> Transferir el sobrenadante a un tubo limpio y ponerlo en hielo. </li><li> Determinar la concentración de proteína en el extracto usando un ensayo de BCA o similar. </li></ol> 4. Congelar los extractos de secado <ol><li> Dependiendo de la masa de proteína requerida (es decir, 100 g por tubo), se diluye el homogeneizado en consecuencia y dispensar alícuotas en un recipiente etiquetado estéril 5,0 ml, Falcon (12x75 mm) tubos. Con frecuencia se utilizan 100 mg / tubo. </li><li> Utilice un 18-20 aguja jeringa estéril para hacer 3 agujeros en la tapa de cada tubo. </li><li> Congelar los tubos ya sea colocándolas en hielo seco durante ~ 10 min o en un congelador -80 ° C durante&gt; 1 h. Conservar a -80 ° C hasta el momento de poner en el liofilizador. </li><li> Conectar el liofilizador y permitir que se equilibre (-100 ° C). Esto tarda aproximadamente 30 min. </li><li> Dependiendo del volumen, el secado por congelación puede ser completado dentro de 3 horas, pero de manera rutinaria dejar las muestras durante la noche. </li><li> Después de la terminación del ciclo de secado, desconectar la bomba de vacío y lentamente permitir que la presión en la cámara. Quite la parrilla. </li><li> En una campana estéril, retire los tapones de los tubos perforados y sustituirlos por otros nuevos (Falcon 352.032). </li><li> Tienda de tubos a -20 ° C. Las muestras se pueden reconstituir en medios de cultivo, o tampones otros, y se utiliza en la función o ensayos bioquímicos. </li></ol> 5. Los resultados representativos La Figura 1 muestra la tinción de una proteínagel cargado con extracto de bazo y de islotes empobrecido tejido pancreático (etiquetado acinar) y se purificó islotes humanos (islotes marcados). Los resultados muestran una buena representación de las proteínas de peso molecular diferente para cada tejido. La capacidad de los extractos de tejido humano para estimular la proliferación de células T se ensayó usando un ensayo de proliferación de CFSE basado en 7 (Figura 2). El PBMC utilizado en este ensayo fueron aisladas de un individuo con diabetes tipo 1. La magnitud de la respuesta se expresa como una relación del número de células CFSE dim por 5.000 CD4 +, células CFSE brillantes sin antígeno: de células CFSE dim por 5.000 CD4 +, células CFSE luminosas con antígeno de 7 muestras por triplicado. Los resultados muestran una débil, pero la proliferación detectable, en respuesta a acinar (CD1 = 3,5) y una respuesta más fuerte con el extracto de islote (6,8). Virus de la influenza inactivado (CDI = 142,6) se incluye como unacontrol positivo. <img alt="Figura 1" src="/files/ftp_upload/4230/4230fig1.jpg" /> Figura 1. Gel de proteína. <img alt="Figura 2" src="/files/ftp_upload/4230/4230fig2.jpg" /> Resultados de la figura 2. De un ensayo de proliferación CFSE basado contra acinar y extracto de islotes. <a href="http://www.jove.com/files/ftp_upload/4230/4230fig2large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> .

<ol>
	<li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+to+measuring+human+islet-antigen+specific+T+cell+function+in+type+1+diabetes.">Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes.</a> Clin. Exp. Immunol. 162, 197-209 (2010).</li><li>Beckhove, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+T+cell-based+identification+of+human+tumor+tissue+antigens+by+automated+two-dimensional+protein+fractionation.">Rapid T cell-based identification of human tumor tissue antigens by automated two-dimensional protein fractionation.</a> J. Clin. Invest. 120, 2230-2242 (2010).</li><li>Mannering, S. I., Brodnicki, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+CD4++T-cell+specificity+and+function+in+Type+1+diabetes.">Recent insights into CD4+ T-cell specificity and function in Type 1 diabetes.</a> Expert Review of Clinical Immunology. 3, 557-564 (2007).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+A-chain+of+insulin+is+a+hot-spot+for+CD4++T+cell+epitopes+in+human+type+1+diabetes.">The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.</a> Clin. Exp. Immunol. 156, 226-231 (2009).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insulin+A-chain+epitope+recognized+by+human+T+cells+is+posttranslationally+modified.">The insulin A-chain epitope recognized by human T cells is posttranslationally modified.</a> J. Exp. Med. 202, 1191-1197 (2005).</li><li>Moon, H. C., Joffe, M., Thomas, H. E., Kay, T. W. H., Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+extracting+tissue+proteins+for+use+in+lymphocyte+function+assays.">A method for extracting tissue proteins for use in lymphocyte function assays.</a> Journal of Immunological Methods. 359, 56-60 (2010).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> J. Immunol. Methods. 283, 173-183 (2003).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+Cell+Proliferation+in+Response+to+GAD+and+Proinsulin+in+Healthy,+Pre-diabetic,+and+Diabetic+Donors.">CD4+ T Cell Proliferation in Response to GAD and Proinsulin in Healthy, Pre-diabetic, and Diabetic Donors.</a> Ann. N.Y. Acad. Sci. 1037, 16-21 (2004).</li><li>Mannering, S. I., Purcell, A. W., Honeyman, M. C., McCluskey, J., Harrison, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T-cells+recognise+N-terminally+Fmoc-modified+peptide.">Human T-cells recognise N-terminally Fmoc-modified peptide.</a> Vaccine. 21, 3638-3646 (2003).</li><li>Peakman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+preparations+of+GAD65,+proinsulin,+and+the+islet+tyrosine+phosphatase+IA-2+for+use+in+detection+of+autoreactive+T-cells+in+type+1+diabetes:+report+of+phase+II+of+the+Second+International+Immunology+of+Diabetes+Society+Workshop+for+Standardization+of+T-cell+assays+in+type+1+diabetes.">Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of autoreactive T-cells in type 1 diabetes: report of phase II of the Second International Immunology of Diabetes Society Workshop for Standardization of T-cell assays in type 1 diabetes.</a> Diabetes. 50, 1749-1754 (2001).</li><li>Honey, K., Rudensky, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+cysteine+proteases+regulate+antigen+presentation.">Lysosomal cysteine proteases regulate antigen presentation.</a> Nat. Rev. Immunol. 3, 472-482 (2003).</li></ol>

No hay conflictos de interés declarado.

Este protocolo fue desarrollado porque queríamos generar un extracto de tejido humano que estaba libre de productos químicos tóxicos como detergentes. Específicamente, se han utilizado para preparar los extractos de tejido humano que se pueden utilizar en ensayos de la función inmune humano in vitro. Los extractos preparados usando este protocolo también se puede reconstituir en cualquier tampón y se utiliza para muchos análisis bioquímicos, tales como la cromatografía de Western Blot o líquido. Esto...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> 5 ml 12 x 75 mm tubos de poliestireno estériles </td><td> BD Falcon </td><td> 352054 </td><td></td></tr><tr><td> Tapones para tubos de tubos de poliestireno (arriba) </td><td> BD Falcon </td><td> 352032 </td><td></td></tr><tr><td> Tubos de 50 ml estériles </td><td> Becton Dickinson </td><td> 352070 </td><td></td></tr><tr><td> Acetonitrilo </td><td> Mallinckradt Químicos </td><td> 2856-10 </td><td></td></tr><tr><td> Butan-1-ol </td><td> Sigma Aldrich </td><td> 537993-IL </td><td></td></tr><tr><td> Homogeneizador: PRO200 </td><td> Bio-estrategia </td><td> 01-01200 </td><td> 10 x 115 mm de diente de sierra generador </td></tr><tr><td> Liofilizador </td><td></td><td></td><td> Virtis, Benchtop 4K </td></tr><tr><td> Aguja estéril de calibre 18-20 </td><td> Becton Dickinson </td><td> REF 302032 </td><td></td></tr><tr><td> CMRL-1066 Medium </td><td> Sigma </td><td> C0422 </td><td></td></tr><tr><td> PBS </td><td> Sigma </td><td> D8537 </td><td></td></tr></tbody></table> Tabla 1. Reactivos y equipos específicos.

extraction of tissue antigens for functional assays

Muchos de los objetivos de antígeno de la respuesta inmune adaptativa, reconocidos por células B y T, no se han definido 1. Esto es particularmente cierto en las enfermedades autoinmunes y el cáncer 2. Nuestro objetivo es investigar los antígenos reconocidos por las células T en la enfermedad de tipo 1 autoinmune diabetes 1,3,4,5. Para analizar humanos respuestas de células T contra el tejido en el que los antígenos reconocidos por las células T no se identifican hemos desarrollado un método para extraer antígenos de proteínas a partir de tejido humano en un formato que es compatible con los ensayos funcionales 6. Respuestas previamente, las células T a los extractos de tejido no purificados no se pudo medir debido a que los métodos de extracción de producir un lisado que contiene detergentes que eran tóxicas para las células humanas mononucleares de sangre periférica. Aquí se describe un protocolo para la extracción de proteínas de los tejidos humanos en un formato que no es tóxico para las células T humanas. El tejido se homogeneizó en una mezcla de butan-1-ol, acetonitrilo y water (BAW). La concentración de proteína en el extracto de tejido se mide y una masa conocida de la proteína se dividió en alícuotas en tubos. Después de la extracción, los disolventes orgánicos se eliminan por liofilización. Extractos de tejidos liofilizadas pueden almacenarse hasta que se necesite. Para uso en ensayos de la función inmunitaria, una suspensión de células inmunes, en medios de cultivo apropiados, se puede añadir directamente al extracto liofilizado. La producción de citocinas y la proliferación de PBMC, en respuesta a los extractos preparados con este método, se mide fácilmente. Por lo tanto, nuestro método permite la rápida preparación de lisados ​​de tejidos humanos que se pueden utilizar como fuente de antígenos en el análisis de las respuestas de células T. Se sugiere que este método facilitará el análisis de la respuesta inmune adaptativa a los tejidos en el trasplante, el cáncer y la autoinmunidad.

Watch this Scientific Journal Video about Extraction of Tissue Antigens for Functional Assays at JoVE.com

Extraction of Tissue Antigens for Functional Assays

Un protocolo simple de preparar los extractos de tejido humano para ser utilizado como una fuente de antígenos funcionales en ensayos de células T se describe. Este método permite respuestas de células T a los antígenos derivadas de tejido a medir<em&gt; In vitro</em&gt;.

Extracción de los antígenos tisulares para Ensayos funcionales

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

extraction-of-tissue-antigens-for-functional-assays

Research

JoVE Journal

Biology

10.8K vistas.  St. Vincent's Institute of Medical Research. Un protocolo simple de preparar los extractos de tejido humano para ser utilizado como una fuente de antígenos funcionales en ensayos de células T se describe. Este método permite respuestas de células T a los antígenos derivadas de tejido a medir In vitro.

Video: Extraction of Tissue Antigens for Functional Assays

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

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Co-sedimentación de ensayo, pues el análisis de proteína de unión a la F-actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Cristalización de las proteínas de membrana para la Determinación de la estructura con mesofases lipídico

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Los ensayos farmacológicos y genéticos funcionales para manipular la regeneración de la planaria Japonica Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Comparación cuantitativa de Cis-Elemento regulador (CRE) Las actividades de transgénicos Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

El uso de un robot para la cristalización de alto rendimiento de proteínas de membrana en mesofases lipídicas

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

Los pequeños extractos nucleares para ensayos funcionales de los mecanismos de expresión de genes

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Los ensayos de viabilidad de las células en cultivo

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Aislamiento y análisis funcional de las mitocondrias de las células cultivadas y ratón de Tejidos

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Preparación de núcleos de tejido incluidas en parafina fijadas con formalina para ambos RNA y DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...