Questo lavoro è stato sostenuto dal Biotechnology and Biological Sciences Research Council (sovvenzione 31/C15982), il Canadian Institutes of Health Research, la Fondazione canadese per l&#39;innovazione, la British Columbia Fondo di conoscenza per lo sviluppo, la Michael Smith Fondazione per la Ricerca sulla Salute (sovvenzione per CJR Loewen), e lotta per la vista.

Materiali e metodi: <ol><li> Ceppi di lievito: <ul><li> Acp1-GFP (GFP:: Il suo): C-terminale GFP-tagging delle Acp1 è stato generato da una PCR-mediata mediante ricombinazione omologa pKT128 plasmide (GFP e contiene HIS5). Trasformanti positivi sono stati confermati mediante microscopia confocale e colonia PCR. Lo sfondo ceppo è stato BY7043 (MAT alfa can1Δ:: STE2pr-lue2 lyp1Δ his3Δ1 leu2Δ0 ura3Δ0 met15Δ0) da Boone laboratorio (Tong e Boone, 2006). </li><li> Cancellazione Array Mutant (DMA) (xxx:: KanR): si tratta di una serie di circa 5.000 mutanti di delezione di geni non essenziali. Tutti questi geni non essenziali sono stati eliminati il ​​G418. Lo sfondo deformazione della matrice è BY4741 (Mata his3_1 leu2_0 met15_0 ura3_0). Il nostro DMA era originario di Boone laboratorio. </li></ul></li><li> Incorporando Acp1-GFP nel DMA: Il protocollo seguito per fare una serie GFP è adattato dal Analisi sintetica genetica (SGA) tecnologia (Tong e Boone, 2006) con modifiche. Tutti i passaggi pinning replica sono fatte utilizzando un rotore Singer ad alta densità arraying sistema robotico. In alternativa, manuale pinning o una base liquida ad alta densità di robot arraying possono essere utilizzati. Acronimi: YPD, estratto-peptone-destrosio lievito, SD, di abbandono di sintesi; LRK, Lue / Arg / Lys abbandono dei media; LHRK, Lue / suo / Arg / Lys abbandono dei media Preparazione <ol><li> Crescere un prato di Acp1-GFP cellule versando a 5 ml di notte Acp1-GFP cultura su un piatto YPD. Lasciare seccare la lastra a sufficienza da una fiamma o in una cappa aspirante biologico. </li><li> Incubare la piastra a 30 ˚ C durante la notte. </li><li> Uso di un array di robot Acp1-GFP versare nel 1536 colonie per piastra. Allo stesso tempo, preparare una nuova copia del DMA replica da appuntare al YPD + piastre G418. </li><li> Incubare le piastre a 30 ˚ C durante la notte. Accoppiamento e selezione diploide </li><li> I-Mate Acp1 GFP ceppo con la DMA di replica pinning e oltre-che le colonie su piastre YPD. </li><li> Incubare le piastre a 30 ˚ C durante la notte. </li><li> Replica piastra le colonie su SD - I suoi piatti G418 + per selezionare le cellule diploidi (Mata / MATalpha). </li><li> Incubare le piastre a 30 ˚ C durante la notte. Sporulationg e germinazione </li><li> Replica piastra le colonie diploide su supporti sporulazione maggiore, che contiene livelli ridotti di fonti di carbonio e di azoto per indurre la formazione di spore meiotico. </li><li> Incubare le piastre a 25 ˚ C per un minimo di 5 giorni. </li><li> Germinare la progenie MATA meiotico di replica-plating le colonie su LRK media e incubare le piastre a 30 ˚ C per due giorni. </li><li> Questo supporto sarà indurre la germinazione delle cellule aploidi dalle spore. Dal momento che un LUE2 quadro di lettura di apertura (ORF) è integrato al MATA specifico promotore STE2, tutti i haploids germinato sarà Mata. Selezione di entrambi gli alleli </li><li> Ora è necessario selezionare sia l&#39;allele GFP e il singolo allele mutante. Per fare questo, le cellule sono MATA replica placcata su LRK + G418 media, che sceglie di cellule aploidi che portano la delezione del gene (xxx:: kanR). </li><li> Incubare le piastre a 30 ˚ C durante la notte. </li><li> Infine, la progenie MATA meiotico sono placcati in replica su LHRK + G418 Media per selezionare per la crescita dei singoli mutanti (xxx:: kanR) anche ospitare Acp1-GFP (GFP:: Il suo). Queste placche possono essere conservati a 4 ˚ C per un massimo di 3 mesi. </li></ol> Il protocollo seguito per fare una serie GFP è adattato dal Analisi sintetica genetica (SGA) tecnologia (Tong e Boone, 2006) con modifiche. Tutti i passaggi pinning replica sono fatte utilizzando un rotore Singer ad alta densità arraying sistema robotico. In alternativa, manuale pinning o una base liquida ad alta densità di robot arraying possono essere utilizzati. Acronimi: YPD, estratto-peptone-destrosio lievito, SD, di abbandono di sintesi; LRK, Lue / Arg / Lys abbandono dei media; LHRK, Lue / suo / Arg / Lys abbandono dei media Preparazione </li><li> Microscopia: <ol><li> Scegli il desiderato mutante (s) che esprime Acp1-GFP direttamente dal piatto. </li><li> Crescere le cellule a 30 ° C in YPD o SD - I suoi mezzi di accedere alla fase precoce. </li><li> Visualizzare al microscopio confocale utilizzando il filterset GFP. Un esempio di risultato atteso è illustrato nella Figura 1. </li></ol></li></ol> SGA media: I media di crescita (YPD) e dei media la selezione (SD) utilizzato in questo protocollo è utilizzato di routine nella biologia molecolare del lievito. Si prega di fare riferimento al metodo di lievito (Amberg et al., 2005) per le descrizioni dettagliate. LRK, LHRK media (per 400 ml totali) <table border="0"><tbody><tr><td> Aggiungi al fine </td><td> Quantità </td></tr><tr><td> Lievito base azotata con solfato di ammonio </td><td> 2,7 g </td></tr><tr><td> Aminoacidomescolare </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% di destrosio </td><td> 40 ml </td></tr><tr><td colspan="2"> Raffreddare 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"> Mescolare, versare 50 ml per piastra </td></tr></tbody></table> LRK, LHRK + G418 media (per 400 ml totali) <table border="0"><tbody><tr><td> Aggiungi al fine </td><td> Quantità </td></tr><tr><td> Lievito base azotata senza solfato di ammonio </td><td> 0,7 g </td></tr><tr><td> Acido mix di aminoacidi </td><td> 0,8 g </td></tr><tr><td> Glutammato monosodico </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% di destrosio </td><td> 40 ml </td></tr><tr><td colspan="2"> Raffreddare 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 di G418 </td><td> 0,4 ml </td></tr><tr><td colspan="2"> Mescolare, versare 50 ml per piastra </td></tr></tbody></table> Arricchito sporulazione media (per 400 ml totali) <table border="0"><tbody><tr><td> Aggiungi al fine </td><td> Quantità </td></tr><tr><td> Acetato di potassio </td><td> 4 g </td></tr><tr><td> Estratto di lievito </td><td> 0,4 g </td></tr><tr><td> Destrosio </td><td> 0,2 g </td></tr><tr><td> Sporulazione mix di aminoacidi </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"> Raffreddare a 65 ˚ C </td></tr><tr><td> 200 mg / ml di G418 </td><td> 0,4 ml </td></tr><tr><td colspan="2"> Mescolare, versare 50 ml per piastra </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>

Questo metodo può aiutare in modo efficiente incorporare un marcatore mitocondriale, Acp1-GFP in vari contesti mutante. Essa si basa sull&#39;utilizzo di un sistema robotico, e può essere facilmente adottata per l&#39;utilizzo con qualsiasi sistema robotico. Questa procedura può essere utilizzata anche per l&#39;integrazione di altri tipi di marcatori. Ad esempio, per visualizzare ER, si usano abitualmente il marcatore Erg11-GFP. Nelle nostre immagini rappresentative, un mutante e un tipo selvaggio con la Acp1 GFP-creatore sono stati visualiz ed al microscopio confocale per studiare la morfologia dei mitocondri. Il mutante ha mostrato sconvolto fenotipo mitocondriale, ed è quindi un buon candidato per ulteriori indagini.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SGA media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<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>
</tr>		</tbody>
		</table>
		</div>

a high-throughput method to globally study the organelle morphology in s. cerevisiae

High-throughput metodi per esaminare la localizzazione di proteine ​​o morfologia organello è uno strumento efficace per studiare le interazioni delle proteine ​​e può aiutare a raggiungere una comprensione completa delle vie molecolari. In Saccharomyces cerevisiae, con lo sviluppo del non essenziali serie gene cancellazione, siamo in grado di studiare a livello globale la morfologia degli organelli diversi, come il reticolo endoplasmatico (ER) e la GFP mitocondri utilizzando (o variante)-marcatori in geni diversi contesti. Tuttavia, i marcatori GFP incorporando in ogni singolo mutante è individualmente un lavoro processo ad alta intensità. Qui, descriviamo una procedura che viene normalmente utilizzato nel nostro laboratorio. Utilizzando un sistema robotico per gestire array ad alta densità di lievito e le tecniche di selezione dei farmaci, siamo in grado di ridurre significativamente il tempo richiesto e migliorare la riproducibilità. In sintesi, attraversiamo un GFP-tagged marcatore mitocondriale (Apc1-GFP) ad un alta densità serie di 4.672 mutanti delezione del gene non essenziale dalla robotica replica pinning. Attraverso la selezione diploide, sporulazione, germinazione e la selezione marcatore duale, abbiamo recuperare entrambi gli alleli. Come risultato, ogni mutante aploide singolo contiene Apc1-GFP incorporato al suo locus genomico. Ora, possiamo studiare la morfologia dei mitocondri in tutti i non essenziali sfondo mutante. Utilizzando questo approccio high-throughput, possiamo comodamente studiare e delineare i percorsi e geni coinvolti nella eredità e la formazione di organelli in un genoma impostazione.

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-fusion proteine ​​sono ampiamente utilizzati per visualizzare organelli mediante microscopia confocale. Tuttavia, lo screening per le mutazioni che influenzano la morfologia degli organelli generalmente richiede mutagenesi individuale e richiede molto tempo. Qui, dimostriamo un metodo per integrare simultaneamente organello-GFP marcatori in quasi 5.000 non essenziali geni nel lievito.

Un high-throughput metodo per studiare la morfologia globale organello in S. cerevisiae

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

a-high-throughput-method-to-globally-study-organelle-morphology-s

Nanyang Technological University

Research

JoVE Journal

Biology

10.3K Views.  University of British Columbia - UBC. GFP-fusion proteine ​​sono ampiamente utilizzati per visualizzare organelli mediante microscopia confocale. Tuttavia, lo screening per le mutazioni che influenzano la morfologia degli organelli generalmente richiede mutagenesi individuale e richiede molto tempo. Qui, dimostriamo un metodo per integrare simultaneamente organello-GFP marcatori in quasi 5.000 non essenziali geni nel lievito.

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.

Test di adesione ed invasione di Agar in S. cerevisiae

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

Ricerca

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.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

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.

L&#39;identificazione di complessi proteici con la proteomica quantitativa 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.

A High-Throughput Metodo per la crioconservazione dello sperma e Zebrafish In Vitro Fecondazione

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 metodo per studiare l&#39;architettura tridimensionale dei genomi.

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 Metodo di ibridazione di caratterizzare gli schemi di espressione di mRNA del mouse fetale basso tratto urogenitale

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

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.

Flusso Cytometry basata Purificazione<em&gt; S. cerevisiae</em&gt; Zigoti

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.

L&#39;analisi citofluorimetrica di bimolecular Complementation Fluorescenza: A High Throughput Metodo quantitativo per studiare l&#39;interazione proteina-proteina

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.

High-throughput Immagine Analisi di sferoidi tumorali: Un software applicativo di facile utilizzo per misurare le dimensioni del Spheroids automaticamente e con precisione

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.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

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