Reconocemos el apoyo de la Ciencias Químicas, Geociencias y Ciencias Biológicas División de la Oficina de Ciencias Básicas de Energía, Oficina de Ciencia, EE.UU. Departamento de Energía (número de adjudicación DE-FG02-91ER20021) y la National Science Foundation (MCB 0948584) (FB). Estamos muy agradecidos a la Sra. Karen Bird para la edición del manuscrito.

1. EMS Tratamiento Semillas de Arabidopsis thaliana se mutagénesis utilizando como agente mutágeno de metano sulfonato de etilo (EMS) de 3,4, lo que induce en el genoma de C a T cambios resultantes en la C / G a T / A mutaciones 5-7. <ol><li> Pesar 0,8 g de semillas de Arabidopsis (aproximadamente 40.000 semillas) que llevan el marcador fluorescente orgánulo (específicamente, en este estudio ssGFPHDEL (secuencia señal-GFP-HDEL tetrapéptido) ha sido utilizado como un marcador ER). </li><li> Transferencia de las semillas en un tubo Falcon de 50 ml, a continuación, añadir 25 ml de agua destilada. </li><li> Añadir 0,2% (v / v) sulfonato de etilo metano. </li><li> Incubar durante 16 horas en la oscilante batidora a velocidad baja. </li><li> Aspirar el líquido y desecharlo en un matraz que contenía 1,0 M de NaOH para inactivar el SME. </li><li> Añadir 25 ml de agua al tubo Falcon que contiene las semillas, muy cerca, e invertir 5 veces para lavar las semillas, espere hasta que todas las semillas que se han asentado, y aspirar el agua y desechar en el Na 1,0 M<html> OH matraz. </li><li> Repetir la etapa de lavado hasta 10 veces. </li><li> Después del lavado final, volver a suspender las semillas en una cantidad mínima de agua. </li><li> Proceda a la esterilización de semillas añadir 25 ml de lejía al 10%, agitar con fuerza durante 30 s. Que las semillas se depositan en el fondo, a continuación, verter el cloro y enjuagar con 25 ml de agua estéril. Vierta el agua estéril y añadir 25 ml de etanol al 70%. Agitar los tubos durante 30 s. Que las semillas se depositan en el fondo. Retirar el etanol y el enjuague con 25 ml de agua estéril. Repetir dos veces el lavado con agua estéril, y luego verter las semillas en una placa Petri de plástico que contiene un papel de filtro 3 MM y dejar secar bajo el capó. Almacenar a 4 ° C durante 2 días. </li><li> Placa de las semillas en ½ MS Phytagel (concentración media de medio Murashige y Skoog), de 150 mm placa de Petri (aproximadamente 250-300 semillas para la placa). </li><li> Crecer semillas M1 en la placa durante 2 semanas, para luego trasplantarlas en el suelo. </li><li> Recoger las semillas de plantas individuales M2 M1 M2 para generar las líneas (1000 líneas independientes). </li></ol>ove_title &quot;2&gt;. Detección confocal de las poblaciones de las categorías M2 y M3 En esta sección se describe la observación de las plantas de semillero con un microscopio confocal o de fluorescencia como se describió anteriormente 8. <ol><li> Sesenta semillas de cada línea M2 son cultivadas por 7 a 10 días en cajas Petri de plástico de ½ MS Phytagel, en el mismo plato también se cultivan plantas EMS-5 sin tratamiento (control). </li><li> De cinco a diez cotiledones están montados con la cara abaxial hacia el objetivo (40X) en un portaobjetos de microscopio y encerrados con un cubreobjetos. </li><li> Cada cotiledón se observa, desde la cortical a la región media, bajo fluorescencia para cualquier alteración de la distribución subcelular del marcador orgánulo. </li><li> Plantas positivos se trasplantan en la tierra y sus semillas se recogen y se proyectó de nuevo para confirmar el fenotipo mutante en la generación M3. </li><li> Quite los contaminantes a través de mutaciones de fondo retrocruzamiento al menos tres veces a un genoma de tipo salvaje, posiblemente, containing el deseado marcador fluorescente orgánulo. <ol><li> De la planta madre, el uso de tijeras o pinzas finas retire silicuas maduras y flores abiertas. </li><li> Eliminar los brotes que son demasiado pequeños del meristema. </li><li> Inserte la punta de un par de pinzas entre los pétalos y los sépalos de abrir un capullo de la flor y eliminar todas las anteras. </li><li> Usando un par de pinzas tome una flor madura abierta de la planta padre y frote las anteras sobre el estigma de la planta de castrado. </li></ol></li></ol> 3. Mapeo En esta sección se describe esencialmente la forma de asignar una mutación recesiva con un protocolo modificado de Borevitz 9 que es más rápido si se compara con los métodos de mapeo tradicionales 10,11. Este enfoque utilizar matrices de alta densidad de oligonucleótidos con la capacidad de detectar polimorfismos numerosas sola característica (SFP) en un solo ensayo 12. Uso de la Arabidopsis Affymetrix GeneChip ATH1 matriz es posibleanalizar los aproximadamente 24.000 genes. Un grupo de individuos F 2 que muestran que la mutación se compara con una piscina de tipo salvaje F 2 plantas recolectadas dentro de la misma población segregante. Entonces, la mutación se asignan en la región donde se enriquece la piscina mutante para los alelos mutantes genotipo y por consiguiente en la misma región de la piscina de tipo salvaje resultará enriquecido para los alelos matrices de tipo salvaje 13. <ol><li> El ADN genómico (3 g) se extrae de la homocigotos Columbia mutante (M3), utilizando una Qiagen DNeasy Plant Mini Kit y se somete a Illumina Genoma Analyzer II (GA II) 14 secuenciación. </li><li> El mutante homocigótico Columbia (M3) se cruza con Landsberg erecta para generar una población de mapeo. </li><li> La asignación se realiza en 70 hasta 100 individuos F2 que muestran el fenotipo aberrante y el mismo número de plantas F2 con un fenotipo de tipo salvaje. </li><li> Recoger de cada planta de un disco de la hoja (0,60 mm) utilizando un punzón. Ladisco de la hoja debe ser recogida de las hojas de la misma edad para asegurarse de que tienen cantidades similares de ADN. Las muestras pueden ser procesadas para la extracción genómico por separado o en grupos. En este caso, es posible recoger muestras 5-10 para cada tubo Eppendorf de modo que al final tendrá menos tubos eppendorf de ADN genómico que tiene que ser extraído. </li><li> MasterPure Planta Hoja de purificación de ADN kit (Epicentro) se utiliza para extraer el ADN genómico. El ADN genómico obtenido de cada muestra se cuantifica con un NanoDrop. </li><li> La misma cantidad de ADN genómico de cada muestra se pone entonces juntos hasta un total de 300 ng (~ 30 l) de ADN de la planta; añadir 60 l solución 2.5X cebadores al azar [125 mM Tris-HCl (pH 6,8), 12,5 mM MgCl2, 25 mM 2-mercaptoetanol, 750 ug / ml cebadores oligodesoxirribonucleótidos (octámeros aleatorios)] (Bioprime kit) y 42 l de agua (volumen final de 132 l). </li><li> Desnaturalizan las autoridades nacionales designadas a&gt; 95 ° C durante 5 a 10 min. </li><li> Dejar enfriar en hielo. </li><li> Para cada denaturojo ADN añadir 15 l de mezcla dNTP 10X con biotina dCTP [1 mM biotina-14-dCTP, 1 mM dCTP, 2 mM de dATP, 2 mM de dGTP, 2 mM dTTP en 10 mM Tris-HCl (pH 7,5), 1 mM Na2EDTA] (Bioprime kit), y completar con la polimerasa Klenow 3 l (kit Bioprime). </li><li> Incubar toda la noche a 25 ° C. </li><li> Al día siguiente, añadir 15 l 3 M NaOAc y 400 mL en frío 100% de etanol y mezclar. </li><li> Incubar a -80 ° C durante 1 hora, luego girar a 20.500 xg durante 15 minutos, retire el sobrenadante y se lava con 500 l de EtOH 75% frío. </li><li> Gira a 20.500 xg durante 10 minutos. </li><li> Gránulos secos de ADN a 37 ° C durante 10 min y resuspende en 100 l de agua. </li><li> Utilizar 5 l para comprobar el rendimiento y calidad en un gel (Figura 2). </li><li> Enviar 95 l de etiquetado de reacción para el tipo salvaje y mutante de Arabidopsis hibridación GeneChip ATH1 del Genoma de la matriz. </li><li> Los arreglos son escaneados y los archivos. CEL obtenidos son analizados utilizando el software R ( <a href="http://www.r-project.org/">http://www.r-project.o</a>rg /). </li><li> Instalar el software R, a continuación, abra el programa y pegar la siguiente cadena: fuente (&quot; <a href="http://bioconductor.org/biocLite.R">http://bioconductor.org/biocLite.R</a> &quot;) biocLite () A continuación, pulse la tecla, este regreso va a instalar los paquetes de Bioconductor estándar ( <a href="http://www.bioconductor.org">http://www.bioconductor.org</a> ). </li><li> En una nueva carpeta en el escritorio, descarga desde el siguiente sitio web ( <a href="http://www.naturalvariation.org/methods">http://www.naturalvariation.org/methods</a> ) los archivos: readcel.R SFP.R Map.R ath1V5.RData ColLerCEL.zip El ColLerCEL.zip archivo debe ser descomprimido y pegar los archivos resultantes en la nueva carpeta. </li><li> Cambiar el nombre de los datos obtenidos a partir de su experimento GeneChip en wildtype.CEL y mutant.CEL. </li><li> Copie ahora los datos de GeneChip (wildtype.CEL ymutant.CEL) en la carpeta. </li><li> Abierto I, a) para Mac: haga clic en &quot;Varios&quot;, y luego haga clic en &quot;Cambiar directorio de trabajo.&quot; b) para PC: haga clic en &quot;Archivo&quot; y luego &quot;Cambiar directorio&quot; </li><li> a) para Mac: Seleccione la carpeta que contiene los archivos. Haga clic en &quot;Espacio de trabajo&quot;, &quot;archivo de espacio de trabajo de carga&quot; y seleccione ath1V5.RData, b) para PC: Seleccione la carpeta que contiene los archivos anteriores, haga clic en &quot;Archivo&quot;, luego &quot;espacio de trabajo de carga&quot; y seleccione ath1V5.RData. </li><li> ReadCEL.R abierto el Bloc de notas y copiar todo el texto de R. </li><li> SFP.R abierto el Bloc de notas y copiar todo el texto de R. </li><li> Map.R abierto el Bloc de notas y copiar el texto completo de R. </li><li> En la ventana de la consola, verá el siguiente mensaje: Cromosoma X limita Mb yz Genes de la búsqueda en TAIR pegar en este enlace 3 <a href="http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y&amp;end=z">http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y</a>Y fin = z </li><li> Copia y pega el enlace en su navegador de Internet. Aparecerá una ventana y pedir para abrir o guardar el archivo, debe seleccionar guardar. Elegir un nombre de archivo y adjuntar la extensión &quot;. Xls&quot;, a continuación, haga clic en Guardar. </li><li> Abra el archivo &quot;. Xls&quot;, donde usted puede encontrar la coordenada para el mutante (el resultado se representa gráficamente en la Figura 3). </li><li> Dentro del área asignada en el Illumina montado lee el genoma mutante identificar las transiciones específicas del SME 4 entre los polimorfismos de nucleótido único. Complementar el fenotipo mutante de la transformación con el gen de tipo salvaje (s), utilizando procedimientos estándar 15. </li></ol> 4. Los resultados representativos La Figura 1 muestra el método utilizado para la identificación de un mutante de la ruta secretora mediante cribado microscopía confocal. La figura 2 muestra una preparación típica de ADN marcado genómico para la hibridación matriz. En la figura 3, un resultado típico se esperaba después de analizar los datos obtenidos a partir de la matriz GeneChip genoma de Arabidopsis ATH1 se presenta. <img alt="Figura 1" src="/files/ftp_upload/3809/3809fig1.jpg" /> Figura 1. Plantas transgénicas de Arabidopsis que expresan ssGFPHDEL (ER marcador) (1) fueron cultivadas para producir suficientes semillas para EMS tratamiento (2). Las semillas tratadas con EMS se sembraron entonces para generar plantas M1 (3). Cada planta M1 representa una línea diferente y las semillas se obtuvieron por separado de cada uno de ellos. Semillas se sembraron en M2 ½ sustrato MS (4) y luego examinados para detectar defectos en la morfología de ER por microscopía confocal (5). Durante la proyección hemos encontrado plantas que conserva la morfología de tipo salvaje ER (6) y las plantas que muestran defectuosos fenotipos ER (7). Estas plantas se cultivaron para obtener la generación M3 y para confirmar el fenotipo (8). ADN genómico de la planta M3 fue utilizado para Solexa Illumina secuenciación (a). La misma plantase utilizó también para cruces con Ler en peso para obtener la población de mapeo F2 (b). <img alt="Figura 2" src="/files/ftp_upload/3809/3809fig2.jpg" /> Figura 2. Reacciones Bioprime etiquetado aleatorios (5 l de 100 l) se cargaron en gel de agarosa al 1%. Lane, una es de un grupo de plantas silvestres F2, y el carril b es de un grupo de mutantes plantas F2. (Marcador es 1 Kb de ADN escalera N3232, de la NEB). <img alt="Figura 3" src="/files/ftp_upload/3809/3809fig3.jpg" /> Figura 3. La figura representa un ejemplo de asignación de Col-0 mutación usando Arabidopsis GeneChip ATH1 Genoma variedad de hibridación. La mutación en este ejemplo se encuentra en el cromosoma 1 delimitado por barras verticales. Las barras horizontales representan los umbrales de detección.

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No tenemos nada que revelar.

Aquí se describe una confocal de microscopía basada en el cribado para la identificación de mutantes endomembrane. Este enfoque se puede extender fácilmente a otros orgánulos de la célula para que determinados marcadores de proteínas fluorescentes están disponibles. La pantalla se basa en la identificación de mutantes que muestran una distribución aberrante del marcador fluorescente o bien en el orgánulo meta o para orgánulos que no se supone que contienen el marcador. Respectivamente, estos mutantes represe...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Etilmetano sulfonato </td><td> Sigma </td><td> M0880 </td></tr><tr><td> NaOH </td><td> JT Baker </td><td> 3722-05 </td></tr><tr><td> Murashige Skoog basal media w vitaminas Gamborg </td><td> Phyto Technolog Laboratorie </td><td> M404 </td></tr><tr><td> Phytagel </td><td> Sigma </td><td> P8169-1Kg </td></tr><tr><td> RNeasy mini kit </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> Maestro de la hoja pura de plantas de purificación de ADN kit </td><td> Epicentro </td><td> MPP92100 </td></tr><tr><td> ADN Bioprime sistema de etiquetado </td><td> Invitrogen </td><td> 18094-011 </td></tr><tr><td> El alcohol 200 prueba </td><td> Decan laboratorios inc. </td><td> 2716 </td></tr><tr><td> NaOAc </td><td> JT Baker </td><td></td></tr><tr><td> Chip de genes de Arabidopsis ATH1amplia del genoma </td><td> Affymetrix </td><td> 900385 </td></tr><tr><td> Tubos Falcon de 50 ml </td><td> Corning </td><td> 430290 </td></tr><tr><td> Tubos Eppendorf de 1,5 ml </td><td></td><td></td></tr><tr><td> De papel de filtro de 90mm </td><td> Whatman </td><td> 1001090 </td></tr><tr><td> Balanza Analítica </td><td> Mettler Toledo AB54-S </td><td> na </td></tr><tr><td> Oscilante (de onda) agitador </td><td> Heidolph POLYMAX 1040 </td><td> na </td></tr><tr><td> Centrifugar </td><td> Eppendorf 5417-R </td><td> na </td></tr></tbody></table>

fluorescence-microscopy screening and next-generation sequencing: useful tools for the identification of genes involved in organelle integrity

Este protocolo describe un microscopio de fluorescencia basada en el cribado de las plántulas de Arabidopsis y se describe cómo asignar las mutaciones recesivas que alteran la distribución subcelular de un marcador específico etiquetado fluorescente en la vía secretora. Arabidopsis es un poderoso modelo biológico para estudios genéticos debido a su tamaño del genoma, tiempo de generación y conservación de los mecanismos moleculares entre reinos. La matriz de genotipado como una aproximación al mapa de la mutación en alternativa al método tradicional, basado en marcadores moleculares, es ventajoso porque es relativamente más rápido y puede permitir la asignación de varios mutantes en un marco de tiempo muy corto. Este método permite la identificación de proteínas que pueden influir en la integridad de cualquier orgánulo en las plantas. Aquí, como un ejemplo, proponemos una pantalla para los genes del mapa importantes para la integridad del retículo endoplasmático (ER). Nuestro enfoque, sin embargo, se puede extender fácilmente a otros orgánulos de las células vegetales(Véase por ejemplo 1,2), y por lo tanto representa un paso importante hacia la comprensión de las bases moleculares que rigen en otras estructuras subcelulares.

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Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

Una misión fundamental de la biología celular es definir los mecanismos que subyacen a la identidad de los orgánulos que hacen que las células eucariotas. Aquí proponemos un método para identificar los genes responsables de la integridad morfológica y funcional de los orgánulos de las plantas mediante microscopía de fluorescencia y la próxima generación de herramientas de secuenciación.

Fluorescencia microscopia de Selección y secuenciación de próxima generación: Herramientas útiles para la identificación de genes implicados en la integridad de los Orgánulos

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

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12.2K vistas.  Michigan State University. Una misión fundamental de la biología celular es definir los mecanismos que subyacen a la identidad de los orgánulos que hacen que las células eucariotas. Aquí proponemos un método para identificar los genes responsables de la integridad morfológica y funcional de los orgánulos de las plantas mediante microscopía de fluorescencia y la próxima generación de herramientas de secuenciación.

Video: Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse de imágenes de mitosis después de la transfección de siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Investigación

Biología

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

El uso de ARN de interferencia mediada estrategia de alimentación para la detección de genes implicados en la regulación del cuerpo Tamaño del nematodo<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

La secuenciación de próxima generación de 16S ARN ribosomal genes Amplicones

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

Dirigida análisis de metilación del ADN mediante secuenciación de próxima generación

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplificación, secuenciación de próxima generación, y Genómica Mapeo de ADN retrovirales sitios de integración

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Interactoma-Seq: Un protocolo para la biblioteca de Domainome construcción, validación y selección de Phage Display y la siguiente secuencia de generación

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

Caracterización de microbioma marca Next-Generation 16S rRNA Amplicon ordenando

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

Análisis de microbiota utilizando PCR de dos pasos y secuenciación genética de rRNA 16S de próxima generación

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

Un protocolo de extracción de ADN de mosquitos a base de perlas magnéticas para la secuenciación de próxima generación

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...