Bu çalışma, Biyoteknoloji ve Biyolojik Bilimler Araştırma Konseyi (hibe 31/C15982), Kanada Sağlık Araştırma Enstitüleri, Kanada, British Columbia Bilgi Kalkınma Fonu, İnovasyon Vakfı Sağlık Araştırma CJR için Michael Smith Vakfı (hibe tarafından desteklenen Loewen) ve Sight için mücadele.

Gereç ve yöntem: <ol><li> Maya suşları: <ul><li> Acp1-GFP (GFP:: His): Acp1 C-terminali, GFP-etiketleme plazmid pKT128 (GFP ve HIS5 içerir) kullanarak PCR-aracılı homolog rekombinasyon tarafından oluşturulan oldu. Pozitif transformants konfokal mikroskopi ve koloni PCR tarafından teyit edildi. Boone laboratuarı (Tong ve Boone, 2006): suşu arka plan BY7043 (STE2pr lue2 lyp1Δ his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 MAT alfa can1Δ). </li><li> Silme Mutant Array (DMA) (xxx:: KanR): gerekli olmayan genlerin yaklaşık 5.000 silme mutant bir dizidir. Tüm bu gerekli olmayan genlerin G418 elendikçe. Dizinin gerginlik arka plan BY4741 (Mata his3_1 leu2_0 met15_0 ura3_0). Bizim DMA orijinal Boone laboratuvarı oldu. </li></ul></li><li> DMA Acp1-GFP ekleme: GFP dizi yapmak için aşağıdaki protokol değişiklikleri ile Sentetik Genetik Analizi (SGA) teknolojisi (Tong ve Boone, 2006) uyarlanmıştır. Tüm çoğaltma çivileme adımları, robotik sistem arraying Şarkıcı ROTOR yüksek yoğunluklu kullanılarak yapılır. Alternatif olarak, manuel çivileme veya sıvı bazlı, yüksek yoğunluklu arraying robot kullanılıyor olabilir. Kısaltmalar: YPD, maya özü-pepton-dekstroz, SD, sentetik okulu terk etme; lrk, Lue / Arg / Lys okulu terk medya; LHRK, Lue / His / Arg / Lys okulu terk medya Hazırlık <ol><li> Acp1-GFP hücrelerinin gecede Acp1 GFP kültürünün bir 5 ml YPD bir plaka üzerine dökerek bir çim büyütün. Plaka alev veya biyolojik bir davlumbaz yeterince kurumasını bekleyin. </li><li> 30 ˚ C gecede plaka inkübe edin. </li><li> Plaka başına 1536 koloniler halinde bir robot, dizi Acp1-GFP gerginlik kullanma. Aynı zamanda, çoğaltma YPD + G418 plakaları iğneleyerek DMA yeni bir kopyasını hazırlayın. </li><li> 30 ˚ C gecede plakaları inkübe edin. Çiftleşme ve diploid seçimi </li><li> DMA ile çoğaltma çivileme ve YPD plakalar üzerinde koloniler döşeme Acp1 GFP gerginlik Mate. </li><li> 30 ˚ C gecede plakaları inkübe edin. </li><li> Diploid hücrelerinde (Mata / MATalpha) seçmek için O&#39;nun + G418 plakaları üzerine SD Replica plakalı koloniler. </li><li> 30 ˚ C gecede plakaları inkübe edin. Sporulationg ve Çimlenme </li><li> Mayotik sporların oluşumunu uyarmak için karbon ve azot kaynakları azalmış seviyeleri içeren gelişmiş sporlanma medya Replica plakalı diploid koloniler. </li><li> 25 ˚ C&#39;de 5 gün en az plakalar inkübe edin. </li><li> Lrk medya üzerine çoğaltma kaplama kolonileri Mata mayotik yavrularıyla filizleneceği ve plakalar 30 ˚ C iki gün kuluçkaya yatmaktadır. </li><li> Bu medya sporlar haploit hücreler çimlenme neden olacaktır. LUE2 açılış okuma kare (ORF) Mata özel STE2 organizatörü entegre olduğundan, çimlenmiş tüm haploids Mata olacak. Her iki allel Seçimi </li><li> Şimdi GFP allel ve tek mutant allel seçmek için gerekli. Bunu başarmak için, Mata hücreleri çoğaltma gen delesyon (xxx:: kanR) taşıyan haploit hücreler için seçer lrk + G418 medya, üzerine kaplama. </li><li> 30 ˚ C gecede plakaları inkübe edin. </li><li> Son olarak, Mata mayotik yavrularıyla çoğaltma kaplama tek mutantlar büyüme (xxx: kanR:) seçmek için LHRK + G418 medya üzerine de Acp1-GFP barındıran (GFP:: His). Bu plakalar, 4 ˚ C, 3 ay kadar saklanabilir. </li></ol> GFP dizi yapmak için aşağıdaki protokol değişiklikleri ile Sentetik Genetik Analizi (SGA) teknolojisi (Tong ve Boone, 2006) uyarlanmıştır. Tüm çoğaltma çivileme adımları, robotik sistem arraying Şarkıcı ROTOR yüksek yoğunluklu kullanılarak yapılır. Alternatif olarak, manuel çivileme veya sıvı bazlı, yüksek yoğunluklu arraying robot kullanılıyor olabilir. Kısaltmalar: YPD, maya özü-pepton-dekstroz, SD, sentetik okulu terk etme; lrk, Lue / Arg / Lys okulu terk medya; LHRK, Lue / His / Arg / Lys okulu terk medya Hazırlık </li><li> Mikroskopi: <ol><li> Plaka doğrudan Acp1-GFP ifade eden istenen mutant (ler) seçin. </li><li> 30 hücreleri büyütün ° C - YPD veya SD erken log fazı olan medya. </li><li> GFP filterset kullanarak konfokal mikroskobu ile gözünüzde canlandırın. Beklenen bir sonuç bir örnek Şekil 1&#39;de gösterilir. </li></ol></li></ol> SGA medya: Bu protokolde kullanılan büyüme ortam (YPD) ve seçme medya (SD) rutin maya moleküler biyoloji kullanılır. Lütfen detaylı açıklamaları için (Amberg ve ark, 2005) Maya Yöntemleri bakın. Lrk, LHRK medya (toplam 400 ml için) <table border="0"><tbody><tr><td> Amacıyla ekle </td><td> Miktar </td></tr><tr><td> Maya amonyum sülfat ile azotlu baz </td><td> 2.7 g </td></tr><tr><td> Amino asitkarışım </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"> Otoklav </td></tr><tr><td> 20% Dekstroz </td><td> 40 ml </td></tr><tr><td colspan="2"> 65 Soğuk ˚ C </td></tr><tr><td> 100 mg / ml canavanine </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, plaka başına 50 ml dökün </td></tr></tbody></table> Lrk, LHRK + G418 medya (toplam 400 ml için) <table border="0"><tbody><tr><td> Amacıyla ekle </td><td> Miktar </td></tr><tr><td> Amonyum sülfat olmadan Maya azotlu baz </td><td> 0.7 g </td></tr><tr><td> Amino asit karışımı </td><td> 0.8 g </td></tr><tr><td> Monosodyum Glutamat </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"> Otoklav </td></tr><tr><td> 20% Dekstroz </td><td> 40 ml </td></tr><tr><td colspan="2"> 65 Soğuk ˚ C </td></tr><tr><td> 100 mg / ml canavanine </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, plaka başına 50 ml dökün </td></tr></tbody></table> Zenginleştirilmiş sporlanma medya (toplam 400 ml için) <table border="0"><tbody><tr><td> Amacıyla ekle </td><td> Miktar </td></tr><tr><td> Potasyum asetat </td><td> 4 g </td></tr><tr><td> Maya özü </td><td> 0.4 g </td></tr><tr><td> Dekstroz </td><td> 0.2 g </td></tr><tr><td> Sporlanma amino asit karışımı </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"> Otoklav </td></tr><tr><td colspan="2"> 65 Soğuk ˚ C </td></tr><tr><td> 200 mg / ml G418 </td><td> 0.4 ml </td></tr><tr><td colspan="2"> Mix, plaka başına 50 ml dökün </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>

Bu yöntem çeşitli mutant arka planla mitokondriyal marker, Acp1-GFP dahil verimli bir şekilde yardımcı olabilir. Robotik bir sistem kullanımına dayanır ve herhangi bir robot sistemi ile kullanmak için kolayca kabul edebilir. Bu işlem aynı zamanda belirteç diğer türleri birleştiren için kullanılabilir. Örneğin, ER görselleştirmek için, rutin marker Erg11-GFP kullanabilirsiniz. Temsili görüntü, bir mutant ve Acp1-GFP yapımcısı ile vahşi bir türü visualiz ed mitokondri morfolojisi çalışma...

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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

Protein lokalizasyon veya organel morfolojisi incelemek için yüksek verimlilik yöntemleri protein etkileşimleri çalışmak için etkili bir araçtır ve moleküler yollarının kapsamlı bir anlayış elde etmenize yardımcı olabilir. Saccharomyces cerevisiae, zorunlu olmayan gen silme dizi gelişme, global olarak endoplazmik retikulum (ER) ve mitokondri kullanarak GFP (veya türevi) belirteçlerinin farklı gen geçmişleri gibi farklı organellerin morfoloji eğitim görebilirsiniz. Ancak, tek tek her mutant içeren GFP işaretleri, ayrı ayrı emek-yoğun bir süreçtir. Burada, bizim laboratuvarda rutin olarak kullanılan bir prosedür açıklanmaktadır. Yüksek yoğunluklu maya dizileri ve ilaç seçimi teknikleri işlemek için bir robot sistemi kullanarak, önemli ölçüde zaman gerekli kısaltmak ve tekrarlanabilirlik geliştirmek. Kısacası, biz robot çoğaltma iğneleyerek 4672 gerekli olmayan gen silme mutant bir yüksek yoğunluklu bir dizi GFP etiketli mitokondriyal bir işaretleyici (Apc1 GFP) kesişir. Diploid seçimi, sporlanma, çimlenme ve çift işaretleyici seçimi sayesinde, her iki allel kurtarabilirsiniz. Sonuç olarak, her haploid tek mutant Apc1-GFP genomik lokustaki dahil içerir. Şimdi, biz, gerekli olmayan tüm mutant arka planda mitokondri morfolojisi eğitim görebilirsiniz. Bu yüksek verimlilik yaklaşımı kullanarak, uygun bir çalışma ve genom bir ortamda miras ve organellerin oluşumu ilgili yollar ve genler tasvir.

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-füzyon proteinleri yaygın konfokal mikroskobi organelleri görselleştirmek için kullanılır. Ancak, genellikle bireysel mutagenez organellerin morfolojisini etkileyen mutasyonlar için tarama gerektirir ve zaman alıcıdır. Burada, neredeyse 5.000 maya gerekli olmayan genlerin aynı anda organel-GFP işaretleri dahil etmek için bir yöntem göstermektedir.

S. cerevisiae &quot;adlı organel morfolojisi küresel çalışma yüksek verimli bir yöntem

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 Görüntüleme.  University of British Columbia - UBC. GFP-füzyon proteinleri yaygın konfokal mikroskobi organelleri görselleştirmek için kullanılır. Ancak, genellikle bireysel mutagenez organellerin morfolojisini etkileyen mutasyonlar için tarama gerektirir ve zaman alıcıdır. Burada, neredeyse 5.000 maya gerekli olmayan genlerin aynı anda organel-GFP işaretleri dahil etmek için bir yöntem göstermektedir.

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.

S. cerevisiae yapışma ve Agar Invasion için Testi

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

Biyoloji

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.

Kertenkeleler Hareket Hız ve Yön Visual Hassasiyet Test

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.

S. cerevisiae, kantitatif proteomik protein kompleksleri tanımlanması

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.

Zebra balığı sperm Kriyoprezervasyon için bir Yüksek Verim Yöntem ve In Vitro Gübreleme

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: genomların Üç boyutlu Mimari Eğitim Bir Yöntem.

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.

Yüksek Verimli In situ</emFetal Fare Alt Ürogenital Yolu mRNA İfade Desenler Karakterize> Hibridizasyon Yöntemi

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.

Yaşa bağımlı Genomik İstikrarsızlık incelenmesi S. cerevisiae Kronolojik ömrü Modeli

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.

Akış Sitometri tabanlı Arıtma<em&gt; S. cerevisiae</em&gt; Zigot

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.

Bimoleküler Floresan tamamlama akışı Sitometrik Analizi: Protein-protein etkileşim Eğitim Bir Yüksek Verimli Kantitatif Yöntemi

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.

Tümör sferoidlerin Yüksek throughput Görüntü Analizi: Otomatik ve Doğru küremsiler boyutunu ölçmek için kullanıcı-dostu Yazılım Uygulama

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