Este trabajo fue apoyado por la Biotecnología y Ciencias Biológicas de Investigación (subvención 31/C15982), los Institutos Canadienses de Investigación en Salud, la Fundación Canadiense para la Innovación, el Conocimiento Columbia Británica Fondo para el Desarrollo, la Fundación Michael Smith de Investigación en Salud (subvención a CJR Loewen), y la lucha por la vista.

Material y métodos: <ol><li> Cepas de levadura: <ul><li> ACP1-GFP (GFP:: Su): C-terminal GFP-etiquetado de ACP1 fue generado por una PCR mediada por recombinación homóloga utilizando el plásmido pKT128 (contiene las buenas prácticas agrarias y HIS5). Transformantes positivos fueron confirmados por microscopía confocal y la colonia PCR. La cepa de antecedentes se BY7043 (MAT alfa can1Δ:: STE2pr-lue2 lyp1Δ his3Δ1 leu2Δ0 ura3Δ0 met15Δ0) de Boone laboratorio (Tong y Boone, 2006). </li><li> Eliminación de matriz mutante (DMA) (xxx:: KanR): es un conjunto de alrededor de 5.000 mutantes de deleción de genes no esenciales. Todos estos genes no esenciales fueron eliminados en la G418. La cepa de antecedentes de la matriz es BY4741 (MATA his3_1 leu2_0 met15_0 ura3_0). Nuestra DMA fue originalmente de Boone laboratorio. </li></ul></li><li> La incorporación de ACP1-GFP en la DMA: El siguiente protocolo para la toma de una amplia GFP es una adaptación del análisis genético sintético (SGA) tecnología (Tong y Boone, 2006) con modificaciones. Todos los pasos están depositadas réplica hecha usando un ROTOR Cantante de alta densidad arraying sistema robótico. Por otra parte, manual o fijando una base líquida de alta densidad de robots arraying puede ser utilizado. Siglas: YPD, extracto de levadura peptona-dextrosa, SD, el abandono de síntesis; LRK, Lue / Arg / Lys deserción medios de comunicación; LHRK, Lue / Su / Arg / Lys medios de deserción escolar Preparación <ol><li> Crecer el césped de ACP1-GFP células mediante el vertido de un 5 ml de la noche ACP1-GFP cultura sobre una placa de YPD. Permitir que la placa se seque lo suficiente por una llama o en una campana de extracción biológica. </li><li> Incubar la placa a 30 ˚ C durante la noche. </li><li> El uso de un robot, matriz ACP1-GFP cepa en 1536 colonias por placa. Al mismo tiempo, preparar una nueva copia de la DMA por réplica depositadas en placas YPD + G418. </li><li> Incubar las placas a 30 ˚ C durante la noche. El apareamiento y la selección diploide </li><li> Compañero de la cepa ACP1-GFP con la DMA por réplica de fijación y colocación de más de las colonias en las placas de YPD. </li><li> Incubar las placas a 30 ˚ C durante la noche. </li><li> Placa de réplica, las colonias en SD - Su + placas G418 para seleccionar las células diploides (MATA / MATalpha). </li><li> Incubar las placas a 30 ˚ C durante la noche. Sporulationg y la germinación </li><li> Placa de réplica, las colonias diploides en los medios de comunicación una mayor esporulación, el cual contiene los niveles de reducción de fuentes de carbono y nitrógeno para inducir la formación de esporas meióticas. </li><li> Las placas se incuban a 25 ˚ C durante un mínimo de 5 días. </li><li> Germinan las progenies MATa meiótica de las colonias en placas replica-LRK los medios de comunicación e incubar las placas a 30 ˚ C durante dos días. </li><li> Este medio se induce la germinación de las células haploides a partir de esporas. Desde un marco de apertura de la lectura LUE2 (ORF) se integra en el promotor MATa específicos STE2, todos los haploides germinado se MATA. Selección de los dos alelos </li><li> Ahora es necesario seleccionar tanto el alelo GFP y el alelo mutante único. Para lograr esto, las células de Mata son réplica chapada en los medios de comunicación LRK + G418, que selecciona las células haploides que llevan el gen de supresión (xxx:: KanR). </li><li> Incubar las placas a 30 ˚ C durante la noche. </li><li> Por último, las progenies MATa meiótica son réplica chapada en LHRK medios G418 + para seleccionar el crecimiento de los mutantes individuales (xxx:: KanR) también alberga ACP1-GFP (GFP:: Su). Estas placas pueden ser almacenadas a 4 ˚ C durante un máximo de 3 meses. </li></ol> El siguiente protocolo para la toma de una amplia GFP es una adaptación del análisis genético sintético (SGA) tecnología (Tong y Boone, 2006) con modificaciones. Todos los pasos están depositadas réplica hecha usando un ROTOR Cantante de alta densidad arraying sistema robótico. Por otra parte, manual o fijando una base líquida de alta densidad de robots arraying puede ser utilizado. Siglas: YPD, extracto de levadura peptona-dextrosa, SD, el abandono de síntesis; LRK, Lue / Arg / Lys deserción medios de comunicación; LHRK, Lue / Su / Arg / Lys medios de deserción escolar Preparación </li><li> Microscopía: <ol><li> Elija el que desee mutante (s) que expresa ACP1-GFP directamente desde la placa. </li><li> Se cultivan las células a 30 ° C en YPD o SD - Sus medios de comunicación para la fase de registro inicial. </li><li> Visualizar por microscopía confocal utilizando el filterset GFP. Un ejemplo del resultado esperado se muestra en la Figura 1. </li></ol></li></ol> SGA medios de comunicación: Los medios de cultivo (YPD) y los medios de selección (SD) que se utiliza en este protocolo se utiliza habitualmente en biología molecular de levaduras. Por favor refiérase a los métodos de la levadura (Amberg et al., 2005) para mayor información. LRK, LHRK los medios de comunicación (para un total de 400 ml) <table border="0"><tbody><tr><td> Añadir el fin </td><td> Cantidad </td></tr><tr><td> Base nitrogenada de levadura con sulfato de amonio </td><td> 2,7 g </td></tr><tr><td> Aminoácidomezcla </td><td> 0,8 g </td></tr><tr><td> Agar </td><td> 8 g </td></tr><tr><td> dH 2 O </td><td> 360 ml </td></tr><tr><td colspan="2"> Autoclave </td></tr><tr><td> 20% de dextrosa </td><td> 40 ml </td></tr><tr><td colspan="2"> Enfriar a 65 ˚ C </td></tr><tr><td> 100 mg / ml canavanina </td><td> 0,2 ml </td></tr><tr><td> 100 mg / ml thialysine </td><td> 0,2 ml </td></tr><tr><td colspan="2"> Mezclar, verter 50 ml por placa </td></tr></tbody></table> LRK, LHRK + G418 medios de comunicación (para un total de 400 ml) <table border="0"><tbody><tr><td> Añadir el fin </td><td> Cantidad </td></tr><tr><td> Base nitrogenada de levadura sin sulfato de amonio </td><td> 0,7 g </td></tr><tr><td> Mezcla de aminoácidos </td><td> 0,8 g </td></tr><tr><td> Glutamato monosódico </td><td> 0.4 </td></tr><tr><td> Agar </td><td> 8 g </td></tr><tr><td> dH 2 O </td><td> 360 ml </td></tr><tr><td colspan="2"> Autoclave </td></tr><tr><td> 20% de dextrosa </td><td> 40 ml </td></tr><tr><td colspan="2"> Enfriar a 65 ˚ C </td></tr><tr><td> 100 mg / ml canavanina </td><td> 0,2 ml </td></tr><tr><td> 100 mg / ml thialysine </td><td> 0,2 ml </td></tr><tr><td> 200 mg / ml G418 </td><td> 0,4 ml </td></tr><tr><td colspan="2"> Mezclar, verter 50 ml por placa </td></tr></tbody></table> Enriquecido esporulación los medios de comunicación (para el total de 400 ml) <table border="0"><tbody><tr><td> Añadir el fin </td><td> Cantidad </td></tr><tr><td> Acetato de potasio </td><td> 4 g </td></tr><tr><td> Extracto de levadura </td><td> 0,4 g </td></tr><tr><td> Dextrosa </td><td> 0,2 g </td></tr><tr><td> Esporulación mezcla de aminoácidos </td><td> 0,04 g </td></tr><tr><td> Agar </td><td> 8 g </td></tr><tr><td> dH 2 O </td><td> 360 ml </td></tr><tr><td colspan="2"> Autoclave </td></tr><tr><td colspan="2"> Enfriar a 65 ˚ C </td></tr><tr><td> 200 mg / ml G418 </td><td> 0,4 ml </td></tr><tr><td colspan="2"> Mezclar, verter 50 ml por placa </td></tr></tbody></table>

<ol>
	<li>Amberg, D. C., Burke, D., Strathern, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual. , (2005).</li><li>Tong, A. H., Boone, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+genetic+array+analysis+in+Saccharomyces+cerevisiae.">Synthetic genetic array analysis in Saccharomyces cerevisiae.</a> Methods Mol Biol. 313, 171-192 (2006).</li></ol>

Este método puede ayudar a incorporar de manera eficiente un marcador mitocondrial, ACP1-GFP en varios mutantes antecedentes. Se basa en el uso de un sistema robótico, y fácilmente se puede adoptar para su uso con cualquier sistema robótico. Este procedimiento puede ser utilizado también para la incorporación de otros tipos de marcadores. Por ejemplo, para visualizar la sala de emergencia, nosotros usamos el marcador Erg11-GFP. En nuestras imágenes representativas, un mutante y un tipo salvaje con el fabricante d...

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<td>The growth media (YPD) and synthetic dropout media (SD) including LRK and LHRK, and enriched sporulation media used in this protocol is routinely used in yeast molecular biology. Please refer to Methods in Yeast (Amberg et al., 2005) for detailed descriptions.</td>
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a high-throughput method to globally study the organelle morphology in s. cerevisiae

Métodos de alto rendimiento para examinar la localización de proteínas o la morfología orgánulo es una herramienta eficaz para el estudio de interacciones de proteínas y puede ayudar a lograr una comprensión integral de las vías moleculares. En Saccharomyces cerevisiae, con el desarrollo del conjunto de genes no esenciales la eliminación, que el mundo puede estudiar la morfología de los diferentes orgánulos como el retículo endoplasmático (ER) y las buenas prácticas agrarias con las mitocondrias (o variantes), los marcadores en los fondos genéticos diferentes. Sin embargo, la incorporación de los marcadores GFP en cada solo mutante individual es un proceso de trabajo intensivo. Aquí se describe un procedimiento que se utiliza habitualmente en nuestro laboratorio. Mediante el uso de un sistema robótico para manejar matrices de alta densidad de levadura y las técnicas de selección de medicamentos, se puede acortar significativamente el tiempo necesario y mejorar la reproducibilidad. En resumen, se cruza un marcador GFP-etiquetados mitocondrial (Apc1-GFP) en una matriz de alta densidad de 4.672 mutantes supresión de genes no esenciales de robótica depositadas réplica. A través de la selección diploide esporulación, la germinación y el marcador de selección dual, recuperamos los dos alelos. Como resultado, cada uno único mutante haploide contiene Apc1-GFP incorporado en su locus genómico. Ahora, podemos estudiar la morfología de las mitocondrias en todos los antecedentes mutante no esenciales. El uso de este enfoque de alto rendimiento, que convenientemente se puede estudiar y delimitar las vías y genes implicados en la herencia y la formación de orgánulos en un ambiente de todo el genoma.

Watch this Scientific Journal Video about A high-throughput method to globally study the organelle morphology in S. cerevisiae at JoVE.com

A high-throughput method to globally study the organelle morphology in S. cerevisiae

De fusión GFP-proteínas son ampliamente utilizados para visualizar los orgánulos por microscopía confocal. Sin embargo, la detección de mutaciones que afectan a la morfología de los orgánulos por lo general requiere de mutagénesis individual y requiere mucho tiempo. En este caso, se demuestra un método para incorporar al mismo tiempo orgánulo-GFP marcadores en casi 5.000 genes no esenciales en la levadura.

Un método de alto rendimiento a nivel mundial para estudiar la morfología orgánulo en S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

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10.3K vistas.  University of British Columbia - UBC. De fusión GFP-proteínas son ampliamente utilizados para visualizar los orgánulos por microscopía confocal. Sin embargo, la detección de mutaciones que afectan a la morfología de los orgánulos por lo general requiere de mutagénesis individual y requiere mucho tiempo. En este caso, se demuestra un método para incorporar al mismo tiempo orgánulo-GFP marcadores en casi 5.000 genes no esenciales en la levadura.

Video: A high-throughput method to globally study the organelle morphology in S. cerevisiae

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

Ensayo de adhesión y la invasión de agar en S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

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Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identificación de los complejos de proteínas con la proteómica cuantitativa en S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

Un método de alto rendimiento para la crioconservación de esperma de pez cebra y In Vitro Fertilización

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hi-C: Un método para estudiar la arquitectura tridimensional de los genomas.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

Un alto rendimiento In situ Método de hibridación para caracterizar los patrones de expresión de ARNm en el ratón Fetal del tracto urogenital inferior

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

El estudio de dependientes de la edad de Inestabilidad Genómica con el S. cerevisiae Cronológica Modelo Longevidad

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Citometría de flujo basado Purificación de<em&gt; S. cerevisiae</em&gt; Los cigotos

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

El análisis por citometría de Complementación fluorescencia Bimolecular: A High Throughput Método cuantitativo para estudiar la interacción proteína-proteína

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

De alto rendimiento de imagen Análisis de esferoides tumorales: una aplicación de software fácil de usar para medir el tamaño de esferoides forma automática y precisa

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...