Diese Arbeit wurde unterstützt von der Biotechnology and Biological Sciences Research Council (Stipendium 31/C15982), der Canadian Institutes of Health Research, der Canada Foundation for Innovation, die British Columbia Knowledge Development Fund, der Michael Smith Foundation for Health Research (Zuschuss zu CJR unterstützt Loewen) und Fight For Sight.

Material und Methoden: <ol><li> Hefestämme: <ul><li> ACP1-GFP (GFP:: His): C-terminale GFP-Markierung von ACP1 wurde durch eine PCR-vermittelte homologe Rekombination unter Verwendung von Plasmid pKT128 (enthält GFP und HIS5) generiert. Positive Transformanten wurden durch konfokale Mikroskopie und Kolonie-PCR bestätigt. Der Stamm Hintergrund war BY7043 (MAT alpha can1Δ: STE2pr-lue2 lyp1Δ his3Δ1 leu2Δ0 ura3Δ0 met15Δ0) aus Boone lab (Tong und Boone, 2006). </li><li> Deletionsmutante Array (DMA) (xxx:: KanR): es ist ein Array von rund 5.000 Deletionsmutanten von nicht-essentiellen Gene. Alle diese essentiellen Gene wurden auf G418 klopfte. Der Stamm Hintergrund des Arrays ist BY4741 (MATa his3_1 leu2_0 met15_0 ura3_0). Unsere DMA wurde ursprünglich aus Boone Labor. </li></ul></li><li> Die Einbeziehung ACP1-GFP in den DMA: Das folgende Protokoll zur Herstellung eines GFP-Array wird von der Synthetic Genetic Analysis (SGA) Technologie (Tong und Boone, 2006) mit Modifikationen angepasst. Alle Replik Pinning Schritte sind mit einem Singer ROTOR high-density Anordnen Robot-System durchgeführt. Alternativ kann die manuelle Pinning oder eine Flüssigkeit-basierte High-Density-Anordnen Roboters verwendet werden. Abkürzungen: YPD, Hefeextrakt-Pepton-Dextrose, SD, synthetische Dropout, LRK, Lue / Arg / Lys-Dropout Medien; LHRK, Lue / His / Arg / Lys-Dropout Medien Vorbereitung <ol><li> Wachsen Sie einen Rasen ACP1-GFP-Zellen durch Eingießen einer 5 ml über Nacht ACP1-GFP Kultur auf eine YPD Platte. Lassen Sie die Platte ausreichend trocknen durch eine Flamme oder in einem biologischen Abzug. </li><li> Die Platte bei 30 ˚ C über Nacht. </li><li> Mit Hilfe eines Roboters, array ACP1-GFP-Stamm auf 1536 Kolonien pro Platte. Zur gleichen Zeit bereiten eine neue Kopie des DMA durch Nachbildung Pinning auf YPD + G418-Platten. </li><li> Die Inkubation bei 30 ˚ C über Nacht. Paarung und diploid Auswahl </li><li> Mate die ACP1-GFP-Stamm mit der DMA von Replik Pinning und über-legen die Kolonien auf YPD-Platten. </li><li> Die Inkubation bei 30 ˚ C über Nacht. </li><li> Replica-Platte die Kolonien auf SD - His + G418-Platten zu wählen diploiden Zellen (MATa / MATalpha). </li><li> Die Inkubation bei 30 ˚ C über Nacht. Sporulationg und Keimung </li><li> Replica-Platte der diploiden Kolonien über eine verstärkte Sporulation Medien, die reduzierte Mengen an Kohlenstoff und Stickstoff-Quellen für die Bildung von meiotischen Sporen induzieren enthält. </li><li> Die Inkubation bei 25 ˚ C für mindestens 5 Tage. </li><li> Keimen die MATa meiotischen Nachkommen von Replika-Plattierung der Kolonien auf LRK Medien und inkubieren Sie die Platten bei 30 ˚ C für zwei Tage. </li><li> Diese Medien werden induzieren die Keimung von haploiden Zellen, die aus Sporen. Da ein LUE2 Eröffnung Leserahmen (ORF) in der Mata-spezifische STE2 Förderer integriert ist, werden alle Haploiden gekeimt MATa werden. Die Auswahl der beiden Allele </li><li> Es ist jetzt notwendig, um sowohl die GFP-Allel und dem einzigen mutierten Allels zu wählen. Um dies zu erreichen, sind MATa Zellen replica-plated auf LRK + G418 Medien, die für die haploiden Zellen, die das Gen-Deletion (xxx:: kanR) tragen wählt. </li><li> Die Inkubation bei 30 ˚ C über Nacht. </li><li> Schließlich werden die MATa meiotischen Nachkommen replica-plated auf LHRK + G418 Medien für das Wachstum der einzelnen Mutanten (xxx:: kanR) wählen auch beherbergen ACP1-GFP (GFP:: His). Diese Platten können bei 4 ˚ C für bis zu 3 Monaten. </li></ol> Das folgende Protokoll zur Herstellung eines GFP-Array wird von der Synthetic Genetic Analysis (SGA) Technologie (Tong und Boone, 2006) mit Modifikationen angepasst. Alle Replik Pinning Schritte sind mit einem Singer ROTOR high-density Anordnen Robot-System durchgeführt. Alternativ kann die manuelle Pinning oder eine Flüssigkeit-basierte High-Density-Anordnen Roboters verwendet werden. Abkürzungen: YPD, Hefeextrakt-Pepton-Dextrose, SD, synthetische Dropout, LRK, Lue / Arg / Lys-Dropout Medien; LHRK, Lue / His / Arg / Lys-Dropout Medien Vorbereitung </li><li> Mikroskopie: <ol><li> Wählen Sie die gewünschte Mutante (s) zum Ausdruck ACP1-GFP direkt von der Platte. </li><li> Wachsen Zellen bei 30 ° C in YPD-oder SD - Seine Medien frühen log-Phase. </li><li> Visualisieren Sie mit Hilfe der konfokalen Mikroskopie mit der GFP Filtersatz. Ein Beispiel für zu erwartende Ergebnis wird in Abbildung 1. </li></ol></li></ol> SGA-Medien: Das Wachstum Medien (YPD) und wählen Sie Medien (SD) in diesem Protokoll verwendet wird routinemäßig in Hefe Molekularbiologie. Bitte beachten Sie Methods in Hefe (Amberg et al., 2005) für detaillierte Beschreibungen. LRK, LHRK Medien (für 400 ml gesamt) <table border="0"><tbody><tr><td> Fügen Sie in Ordnung </td><td> Betrag </td></tr><tr><td> Hefe Stickstoffbase mit Ammoniumsulfat </td><td> 2,7 g </td></tr><tr><td> AminosäureMischung </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% Dextrose </td><td> 40 ml </td></tr><tr><td colspan="2"> Abkühlen auf 65 ˚ C </td></tr><tr><td> 100 mg / ml Canavanin </td><td> 0,2 ml </td></tr><tr><td> 100 mg / ml thialysine </td><td> 0,2 ml </td></tr><tr><td colspan="2"> Mix, pour 50 ml pro Platte </td></tr></tbody></table> LRK, LHRK + G418 Media (für 400 ml gesamt) <table border="0"><tbody><tr><td> Fügen Sie in Ordnung </td><td> Betrag </td></tr><tr><td> Hefe Stickstoffbase ohne Ammoniumsulfat </td><td> 0,7 g </td></tr><tr><td> Aminosäure-Mix </td><td> 0,8 g </td></tr><tr><td> Mononatriumglutamat </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% Dextrose </td><td> 40 ml </td></tr><tr><td colspan="2"> Abkühlen auf 65 ˚ C </td></tr><tr><td> 100 mg / ml Canavanin </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"> Mix, pour 50 ml pro Platte </td></tr></tbody></table> Angereichert Sporulation Medien (für 400 ml gesamt) <table border="0"><tbody><tr><td> Fügen Sie in Ordnung </td><td> Betrag </td></tr><tr><td> Kaliumacetat </td><td> 4 g </td></tr><tr><td> Hefeextrakt </td><td> 0,4 g </td></tr><tr><td> Traubenzucker </td><td> 0,2 g </td></tr><tr><td> Sporulation Aminosäure-Mix </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"> Abkühlen auf 65 ˚ C </td></tr><tr><td> 200 mg / ml G418 </td><td> 0,4 ml </td></tr><tr><td colspan="2"> Mix, pour 50 ml pro Platte </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>

Diese Methode kann effektiv helfen enthalten eine mitochondriale Marker, ACP1-GFP in verschiedenen Mutanten Hintergrund. Es beruht auf der Verwendung eines Robot-System, und kann leicht für die Verwendung mit jedem Roboter-System übernommen. Dieses Verfahren kann auch für die Einbeziehung anderer Arten von Markierungen verwendet werden. Zum Beispiel, zu visualisieren ER, verwenden wir routinemäßig die Markierung ERG11-GFP. In unserer repräsentativen Bilder, ein mutiertes und ein Wildtyp mit der ACP1-GFP-Hersteller...

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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

High-Throughput-Methoden, um Protein-Lokalisierung oder Organellen Morphologie zu untersuchen ist ein effektives Werkzeug zur Untersuchung der Protein-Wechselwirkungen und kann dazu beitragen, einen umfassenden Verständnis der molekularen Mechanismen. In Saccharomyces cerevisiae, mit der Entwicklung der non-essential Gendeletion Array, können wir global Studie die Morphologie der verschiedenen Organellen wie das endoplasmatische Retikulum (ER) und die Mitochondrien mit GFP (oder eine Variante)-Marker in verschiedenen Gen-Hintergründen. Allerdings Einbeziehung GFP Marker in jeder einzelnen Mutanten individuell ist ein arbeitsintensiver Prozess. Hier beschreiben wir ein Verfahren, das routinemäßig in unserem Labor verwendet wird. Durch den Einsatz eines Roboters zum High-Density-Hefe-Arrays und Drogen Auswahl Techniken umgehen, können wir deutlich verkürzen den Zeitaufwand und erhöhen die Reproduzierbarkeit. In kurzen, überqueren wir eine GFP-markierten mitochondrialen Marker (APC1-GFP), um ein High-Density Array von 4.672 essentielle Gen-Deletionsmutanten durch Robot-Replik Pinning. Durch diploid Auswahl, Sporulation, Keimung und Dual-Marker Auswahl, gewinnen wir beide Allele. Als Ergebnis enthält jeder haploid einzigen mutierten APC1-GFP in ihrer genomischen Locus integriert. Jetzt können wir untersuchen die Morphologie der Mitochondrien in alle nicht unbedingt mutierte Hintergrund. Mit diesem High-Throughput-Ansatz können wir bequem studieren und beschreiben die Wege und Gene in der Vererbung und der Bildung von Organellen in einer genomweiten Einstellung beteiligt.

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-Fusionsproteine ​​sind weit verbreitet, um Organellen durch konfokale Mikroskopie sichtbar zu machen. Allerdings Screening auf Mutationen, die die Morphologie der Organellen beeinflussen erfordert in der Regel individuelle Mutagenese und zeitaufwändig ist. Hier zeigen wir eine Methode, um gleichzeitig aufzunehmen Organellen-GFP-Marker in fast 5.000 Nicht-essentielle Gene in Hefe.

Ein High-Throughput-Verfahren zur global Studie der Organellen-Morphologie in 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 Aufrufe.  University of British Columbia - UBC. GFP-Fusionsproteine ​​sind weit verbreitet, um Organellen durch konfokale Mikroskopie sichtbar zu machen. Allerdings Screening auf Mutationen, die die Morphologie der Organellen beeinflussen erfordert in der Regel individuelle Mutagenese und zeitaufwändig ist. Hier zeigen wir eine Methode, um gleichzeitig aufzunehmen Organellen-GFP-Marker in fast 5.000 Nicht-essentielle Gene in Hefe.

Serie: 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.

Assay für Adhäsion und 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, ...

Forschung

Biologie

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.

Identifizierung von Protein-Komplexen mit der quantitativen Proteomik in 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.

Ein High-Throughput-Methode für Zebrabärblinge Sperma Kryokonservierung und In Vitro Fertilisation

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.

Hallo-C: eine Methode, um die dreidimensionale Architektur des Genoms Study.

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.

Ein hoher Durchsatz In situ Hybridisierung Methode zur mRNA Expression Patterns in der Fetal-Maus unteren Urogenitaltrakts Charakterisieren

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.

Studieren Altersabhängige genomische Instabilität mit der S. cerevisiae Chronologisch Lebensdauer Modell

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.

Durchflusszytometrie-basierte Reinigung von<em&gt; S. cerevisiae</em&gt; Zygotes

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.

Durchflusszytometrieanalyse bimolekularer Fluorescence Komplementierung: A High Throughput Quantitative Methode zur Protein-Protein Interaction Study

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.

Hochdurchsatz-Analyse von Bild Tumorsphäroide: Eine benutzerfreundliche Software-Anwendung, um die Größe der Sphäroide automatisch und präzise messen

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

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.

Hochdurchsatz-Screening für Chemische Modulatoren der Post-transkriptionell regulierten Genen

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

Methods Article

Ein High-Throughput-Verfahren zur global Studie der Organellen-Morphologie in S. cerevisiae