Este trabalho foi financiado pela Biotecnologia e Ciências Biológicas Research Council (concessão 31/C15982), os Institutos Canadenses de Pesquisa em Saúde, a Fundação Canadense para Inovação, a British Columbia Fundo de Desenvolvimento do Conhecimento, a Fundação Michael Smith para Pesquisa em Saúde (conceder a CJR Loewen) e Luta em Prol da Visão.

Materiais e métodos: <ol><li> Cepas de leveduras: <ul><li> ACP1-GFP (GFP:: Seu): C-terminal GFP-tagging de ACP1 foi gerado por uma PCR mediada por recombinação homóloga usando pKT128 plasmídeo (contém GFP e HIS5). Transformantes positivos foram confirmados por microscopia confocal e colônia PCR. O pano de fundo a tensão foi BY7043 (MAT alfa can1Δ:: STE2pr-lue2 lyp1Δ his3Δ1 leu2Δ0 ura3Δ0 met15Δ0) de Boone laboratório (Tong e Boone, 2006). </li><li> Exclusão da Matriz Mutant (DMA) (xxx:: KanR): é um conjunto de cerca de 5.000 mutantes de deleção de genes não-essenciais. Todos estes genes não-essenciais foram eliminados na G418. O pano de fundo a tensão da matriz é BY4741 (MATA his3_1 leu2_0 met15_0 ura3_0). Nosso DMA era originalmente de Boone laboratório. </li></ul></li><li> Incorporando ACP1 GFP-no DMA: O seguinte protocolo para fazer uma série GFP é uma adaptação da Análise genético sintético (SGA) tecnologia (Tong e Boone, 2006) com modificações. Todas as etapas são feitas réplicas pinagem usando um ROTOR Cantor de alta densidade arraying sistema robótico. Alternativamente, manual pinagem ou um líquido baseado em robô arraying de alta densidade podem ser usados. Siglas: YPD, extrair-peptona-dextrose levedura; SD, sintéticos abandono; LRK, Lue / Arg / Lys abandono media; LHRK, Lue / His / Arg / Lys abandono media Preparação <ol><li> Crescer um gramado de ACP1-GFP células derramando um de 5 ml da cultura ACP1 GFP-noite em uma placa YPD. Permitem que o prato para secar suficientemente por uma chama ou um capuz biológica de fumos. </li><li> Incubar as placas a 30 ˚ C durante a noite. </li><li> Usando um robô array, ACP1 GFP-tensão em 1.536 colônias por placa. Ao mesmo tempo, preparar uma nova cópia da DMA pela réplica pinagem para placas YPD + G418. </li><li> Incubar as placas a 30 ˚ C durante a noite. Acasalamento e seleção de diplóides </li><li> Companheiro da estirpe ACP1 GFP com o DMA pela réplica pinagem e mais-que estabelece as colônias em placas YPD. </li><li> Incubar as placas a 30 ˚ C durante a noite. </li><li> Réplica da placa-colônias para SD - Seu placas G418 + para selecionar células diplóides (MATA / MATalpha). </li><li> Incubar as placas a 30 ˚ C durante a noite. Sporulationg e germinação </li><li> Réplica da placa-colônias diplóides em mídia esporulação melhorada, que contém níveis reduzidos de fontes de carbono e nitrogênio para induzir a formação de esporos meióticos. </li><li> Incubar as placas a 25 ˚ C durante um mínimo de 5 dias. </li><li> Germinar as progênies MATA meiótica por replica-plating as colônias em LRK media e incubar as placas a 30 ˚ C por dois dias. </li><li> Esta mídia vai induzir a germinação das células haplóides a partir de esporos. Uma vez que um quadro de leitura LUE2 abertura (ORF) está integrado no promotor STE2 MATA-específica, todos os haplóides serão germinadas Mata. Seleção de ambos os alelos </li><li> Agora é necessário selecionar ambos os alelos do GFP eo único alelo mutante. Para conseguir isso, as células Mata são réplicas banhados em LRK G418 + media, que seleciona para as células haplóides que carregam a deleção do gene (xxx:: kanR). </li><li> Incubar as placas a 30 ˚ C durante a noite. </li><li> Finalmente, as progênies MATA meiótica são réplicas banhados em LHRK G418 + media para selecionar para o crescimento de mutantes simples (xxx:: kanR) também abrigar ACP1-GFP (GFP:: His). Estas placas podem ser armazenadas a 4 ˚ C por até 3 meses. </li></ol> O seguinte protocolo para fazer uma série GFP é uma adaptação da Análise genético sintético (SGA) tecnologia (Tong e Boone, 2006) com modificações. Todas as etapas são feitas réplicas pinagem usando um ROTOR Cantor de alta densidade arraying sistema robótico. Alternativamente, manual pinagem ou um líquido baseado em robô arraying de alta densidade podem ser usados. Siglas: YPD, extrair-peptona-dextrose levedura; SD, sintéticos abandono; LRK, Lue / Arg / Lys abandono media; LHRK, Lue / His / Arg / Lys abandono media Preparação </li><li> Microscopia: <ol><li> Escolha o mutante desejado (s) expressando GFP-ACP1 diretamente do prato. </li><li> Cultivar células a 30 ° C em YPD ou SD - Sua mídia a fase log cedo. </li><li> Visualize por microscopia confocal usando o filterset GFP. Um exemplo de resultado esperado é mostrado na Figura 1. </li></ol></li></ol> SGA mídia: A mídia de crescimento (YPD) e meios de comunicação selecionando (SD) utilizado neste protocolo é utilizado rotineiramente em biologia molecular de leveduras. Por favor, consulte Métodos de levedura (Amberg et al., 2005) para descrições detalhadas. LRK, LHRK mídia (para 400 total de ml) <table border="0"><tbody><tr><td> Adicionar a fim </td><td> Quantidade </td></tr><tr><td> Base de levedura nitrogenadas com sulfato de amônio </td><td> 2,7 g </td></tr><tr><td> Aminoácidomistura </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> Dextrose 20% </td><td> 40 ml </td></tr><tr><td colspan="2"> Arrefecer 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"> Misture, despeje 50 ml por placa </td></tr></tbody></table> LRK, LHRK G418 + media (para 400 total de ml) <table border="0"><tbody><tr><td> Adicionar a fim </td><td> Quantidade </td></tr><tr><td> Base de levedura nitrogenados, sem sulfato de amônio </td><td> 0,7 g </td></tr><tr><td> Mix de aminoácidos </td><td> 0,8 g </td></tr><tr><td> O glutamato monossódico </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> Dextrose 20% </td><td> 40 ml </td></tr><tr><td colspan="2"> Arrefecer 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"> Misture, despeje 50 ml por placa </td></tr></tbody></table> Enriquecido esporulação de mídia (para 400 total de ml) <table border="0"><tbody><tr><td> Adicionar a fim </td><td> Quantidade </td></tr><tr><td> Acetato de potássio </td><td> 4 g </td></tr><tr><td> Extrato de levedura </td><td> 0,4 g </td></tr><tr><td> Dextrose </td><td> 0,2 g </td></tr><tr><td> Esporulação mix 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"> Arrefecer a 65 ˚ C </td></tr><tr><td> 200 mg / ml G418 </td><td> 0,4 ml </td></tr><tr><td colspan="2"> Misture, despeje 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 pode ajudar eficientemente incorporar um marcador mitocondrial, ACP1-GFP em várias formações mutantes. Ele se baseia na utilização de um sistema robótico, e pode facilmente adotado para uso com qualquer sistema robótico. Este procedimento pode ser usado também para incorporar outros tipos de marcadores. Por exemplo, para visualizar ER, que utilizam rotineiramente o marcador Erg11-GFP. No nosso imagens representativas, uma mutante e um tipo selvagem com o fabricante ACP1-GFP foram visualiz ed por mic...

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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

Alto rendimento métodos para examinar localização de proteínas ou morfologia organela é uma ferramenta eficaz para o estudo de interações de proteínas e podem ajudar a alcançar uma compreensão abrangente de vias moleculares. Em Saccharomyces cerevisiae, com o desenvolvimento da matriz de exclusão não essenciais gene, podemos globalmente estudar a morfologia das organelas diferentes, como o retículo endoplasmático (ER) e da mitocôndria utilizando GFP (ou variante) marcadores no gene backgrounds diferentes. No entanto, os marcadores de GFP incorporando em cada mutante único individualmente é um processo trabalhoso. Aqui, descrevemos um procedimento que é utilizado rotineiramente em nosso laboratório. Usando um sistema robótico para lidar com arrays de alta densidade fermento e técnicas de seleção de drogas, podemos reduzir significativamente o tempo necessário e melhorar a reprodutibilidade. Em resumo, nós cruzamos um marcador GFP-tagged mitocondrial (Apc1-GFP) para um conjunto de alta densidade de 4.672 mutantes de deleção do gene não essencial por robótico réplica pinagem. Através da seleção diplóide, esporulação, germinação e seleção marcador dual, nós recuperamos ambos os alelos. Como resultado, cada mutante haplóide única contém Apc1 GFP-incorporados em seu locus genômica. Agora, podemos estudar a morfologia das mitocôndrias em todas fundo não essenciais mutante. Usando essa abordagem de alto rendimento, podemos convenientemente estudar e delinear os caminhos e os genes envolvidos na herança ea formação de organelas em um cenário de todo o 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

GFP-fusão proteínas são amplamente utilizados para visualizar organelas por microscopia confocal. No entanto, a triagem para mutações que afetam a morfologia das organelas geralmente requer mutagênese individual e é demorado. Aqui, nós demonstramos um método para incorporar simultaneamente organela-GFP marcadores em quase 5.000 genes não-essenciais no fermento.

Um método de alto rendimento para estudar a morfologia global organela em 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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Biology

10.3K visualizações.  University of British Columbia - UBC. GFP-fusão proteínas são amplamente utilizados para visualizar organelas por microscopia confocal. No entanto, a triagem para mutações que afetam a morfologia das organelas geralmente requer mutagênese individual e é demorado. Aqui, nós demonstramos um método para incorporar simultaneamente organela-GFP marcadores em quase 5.000 genes não-essenciais no fermento.

Vídeo: 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.

Ensaio para a Adesão e Invasão Agar em S. cerevisiae

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

Biologia

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.

Teste de sensibilidade visual para a velocidade e direção do movimento em 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.

Identificação de complexos de proteínas com proteômica quantitativa em 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.

Um método de alta taxa de transferência para Zebrafish criopreservação de espermatozóides e In Vitro Fertilização

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.

Oi-C: um método para estudar a arquitetura tridimensional de 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.

A High Throughput In situ Método de hibridização para caracterizar padrões de expressão de mRNA no Rato Fetal Trato 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.

Estudando Idade-dependente Instabilidade Genômica usando o S. cerevisiae Modelo Lifespan cronológica

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.

Purificação Citometria de Fluxo com base em<em&gt; S. cerevisiae</em&gt; Zigotos

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.

Citometria de fluxo e de Complementação de fluorescência Bimolecular: A alta taxa de transferência método quantitativo para estudo de interação 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 alta capacidade Análise de Imagem de esferóides tumorais: uma aplicação de software de fácil utilização para medir o tamanho de esferóides automaticamente e com precisão

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