Araştırma NIH 1R15GM094770 tarafından finanse edildi.

1. Colony İzolasyon ve Fiksasyon <ol><li> Belirtilen süre için agar 300 kolonileri (izole edilmiş bir koloni çapı 1-2 mm olmalıdır) inkübe edin. </li><li> Koloni (yüz yukarı) ve dar bir spatula ile altta yatan orta çıkarın. </li><li> % 2 agar birkaç damla koyun 42 ° C katılaşır önce 1 ml agar yüzünde Pipetman ucu ve hemen yer koloni kullanarak bir mikroskop lamı üzerine. </li><li> 42 ° C kolonisi üzerinde% 2 agar birkaç damla koyun ve sağlamlaştırmak için izin. </li><li> Bu protokolün kalan bütün adımlar için eldiven giyin </li><li> Trim jilet ile, 4 blok ve 7 gün boyunca,% 2 paraformaldehid /% 2 glutaraldehid fiksatif içeren bir 3.5 mL borosilikat vida kapaklı flakon koyun ° C </li></ol> 2. Yıkama ve Osmium Tedavi <ol><li> Yaklaşık 1 hafta sonra, 5 dakika sonra koloni (yaklaşık 1.5 ml) iki kez daha tamamen aynı hacmi ile karşılamak için yeterli 0.15m sodyum cacodylate (pH 7.2) ile iki kez 15 dakika boyunca inkübe kimyasal davlumbaz altında buz üzerinde agar blokları yıkamak 1X OS tamponu (100 mM KH 2 PO 4 10 mM MgCl 2, pH 6.0). </li><li> Sonra 1 saat süreyle kimyasal davlumbaz altında agar blokları karşılamak için aynı hacimde% 1 OsO 4 [2 mL OsO 4 (% 2) + 2 ml 2x OS Tampon] şişeleri (buz) aynı miktarda iki kez yıkayın her biri için 10 dakika 1X OS tampon. </li><li> Şişeleri, 4 gece bekletin ° C 1X OS tampon. </li><li> Sabitleme ve bu adımda bütün yıkar tehlikeli atık olarak bertaraf edilir. OsO 4 içeren Tüm pipetler, bitkisel yağ ile 3X durulanır . </li></ol> 3. Yıkama ve dehidratasyon <ol><li> Pasteur pipeti kullanarak her bir 10 dakika soğuk su yukarıdaki gibi aynı hacimde iki kez buz üzerinde Ertesi sabah yıkama agar blokları. </li><li> Sırayla aynı hacimde% 25,% 50,% 75, her 10 dakika, 10 dakika boyunca% 100 etanol ile 2 yıkar takip için% 95 etanol ile yıkayın. 4 gece bekletin ° C% 100 etanol. </li><li> Bu adımda tüm yıkar tehlikeli atık olarak bertaraf edilir. </li></ol> 4. Spurr Kullanıcı Hazırlık <ol><li> Ertesi sabah (mümkün olduğunca erken) Spurr reaktifi hazırlamak için bir davlumbaz altında tek kullanımlık plastik behere aşağıdaki çözümleri (EM Bilimler) tartın. Bu reaktif kullanılarak sonraki tüm iş için, bir davlumbaz kullanmaya devam. (ERL 4221 çözüm 5 gram) (DER 736 çözümü, 4 gram) (NSA çözüm 13 gram) </li><li> bir heyecan çubuğunu kullanarak kaput 20 dakika boyunca bu çözümler karıştırmayın (yavaş) </li><li> DMAE çözüm 0.15 gram ekleyin ve yukarıdaki gibi 20 dakika karıştırın. </li><li> Degas kaputu 1-2 saat için yukarıdaki karışımı. </li></ol> 5. Spurr Kullanıcı Sızma <ol><li> Spurr aynı hacmi ile% 100 her biri için 10 dakika oda sıcaklığında etanol yukarıda agar blokları 5 kez yıkayın kısa sürede yapılır. Son etanol yıkama sonra kalan etanol sadece agar blokları kapsar, böylece Pasteur pipeti kullanarak etanol çıkarın. </li><li> Spurr reaktifi (yaklaşık 0,5 ml) yaklaşık eşit miktarda ekleyin ve tekerlek şişeleri döndürmek ve oda sıcaklığında 15 dakika sonra 30 dakika süreyle bekletilmesi. 30 dakika tamamen Pasteur pipeti kullanarak çözüm kaldırdıktan sonra, ekle Spurr agar bloğu (yaklaşık 1.5 ml) kapsayacak şekilde, oda sıcaklığında 15 dakika boyunca tekerlek şişeleri döndürmek ve 30 dakika bekletilmesi. Spurr yerine, döndürme ve inkübasyon 3 kez daha tekrarlayın. </li><li> Son 30 dakika sonra, Spurr reaktifi kaldırmak ve blokları karşılamak için taze Spurr ekleyin. Agar bloklar 4 saat oda sıcaklığında bekletin. </li><li> Spurr değiştirin ve bir gecede döndürmek. </li><li> Sabah Spurr Kullanıcı değiştirin ve şu güne kadar dönen bırakın. </li><li> Ertesi sabah yer Spurr sonra kalıp alt kapağı kalıpları agar blokları eklemek sayılı silikon kalıplar (Fisher Scientific). 4 saat boyunca 60 ° C&#39;de inkübe </li><li> Daha Spurr ve inkübe 60 ° C gecede kalıpları üstlük 4 saat sonra,. </li><li> Kalıpları blokları çıkardıktan sonra bloklar hala biraz esnek iseniz, kalıpları dönmek ve ek bir 2 gün inkübe </li></ol> 6. Kesit <ol><li> Leica Ultracut S mikrotom ışık mikroskobu ve 0.1 μ iletim EM için ince bölümleri için 0.5 μ kalınlığında kesitler kesmek için kullanılır oldu. </li><li> Kesitler olduğu gibi ışık mikroskobu için, dH 2 O bir damla yerleştirildi, ve 52 ° C ısı blok üzerinde kurutulur. Kurutulmuş bölümlerde daha sonra% 1,0 oranında toluidin mavi, 5-15 saniye için% 1 Sodyum Borat bir düşüş ile boyandı. Boyama sonra, bölümleri, hemen bir su akışı altında yıkanıp kurutulur, montaj medya (KPL) kaplı ve bir kapak kayma örnek üzerinde mühürlenir. </li></ol> Maya ve diğer mikroorganizmaları desen oluşumu ölçme geniş önemi daha önce 1 incelendi. Özellikle not düzensiz topluluklar göre organize mikrobiyal kolonilerin artan işlevselliği. Açıklanan yöntemi, daha büyük uzmanlaşmış koloniler 2 gömmek için bir yöntem bir değişiklik ve modifikasyonlar kısa bir açıklama 3 yayımlanmıştır . Bir örnek, vahşi S. bir koloni merkezinden geçerek bir bölümü hafif bir mikrografı cerevisiae maya koloni Şekil 1&#39;de gösterilmiştir. Aşçı, psödohif ve ovoid maya kolayca ayırt edilebilir ve altta yatan agar işgalci koloni bölgesi de açıktır. Maya bir laboratuvar suşu (W303 arka plan) bir elektron mikrografı bir örneği Şekil 2&#39;de gösterilmiştir. Görüntü yüksek frekanslı sporulated hücreleri içeren koloninin bir bölge. <img src="/files/ftp_upload/2510/2510fig1.jpg" alt="Şekil 1" /> Şekil 1 vahşi bir maya koloni Işık mikroskobu. Bu maya (YPS133) ağaç eksuda 4 izole edilmiştir. 6 gün önce kesit YNA orta 5 koloni inkübe edildi. Açık oklar dolu ok uçları uzamış hücrelerinin zincirleri (psödohif) ve asci göstermektedir dolu oklar temsilcisi oval vejetatif hücreler gösterir, temsilcisi asci göstermektedir. <img src="/files/ftp_upload/2510/2510fig2.jpg" alt="Şekil 2" /> Şekil 2 Elektron mikroskobu bir laboratuvar maya suşu. SH1020 (W303 arka plan) koloni 3 kesit YNA orta 6 gün önce inkübe edildi. Resim asci bir yüksek frekans bölümünde bir bölge gösterir. Bir ok spor duvarın iki katmanlı bir yapı gösterir. <img src="/files/ftp_upload/2510/2510fig3.jpg" alt="Şekil 3" /> Şekil 3. Koloniler halinde sporulated hücrelerinin dağılımı Niceleme: A), uzun kenarı boyunca yığılmış 15 bitişik dikdörtgenler içeren bir ızgara, bir koloni bölümünde bir görüntü merkezi bölgeye bindirilir . Sadece oranları sabit tutarken ızgara sadece koloninin alt ve üst kapakları, ölçeklenir. (B &amp; C) o dikdörtgenin içinde toplam hücrelere göre her dikdörtgen sporulated hücreler kısmını 4 gün eski (B) ve 8-günlük eski (C) koloniler görüntüleri görsel muayene ile belirlenir. Örneğin, koloninin üst kısmında dikdörtgen (&quot;1&quot; etiketli) grafiğin sol tarafına karşılık gelir. 4 koloni ortalaması gösterilir; hata çubukları std gösterilecek. hata.

<ol>
	<li>Kimmel, A. R., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+symmetries:+regulation+of+Dictyostelium+development+through+chemoattractant+and+morphogen+signal-response.">Breaking symmetries: regulation of Dictyostelium development through chemoattractant and morphogen signal-response.</a> Curr Opin Genet Dev. 14, 540-540 (2004).</li><li>Palkova, Z., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+within+a+community:+benefit+to+yeast+long-term+survival.">Life within a community: benefit to yeast long-term survival.</a> FEMS Microbiol Rev. 30, 806-806 (2006).</li><li>Shapiro, J., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significances+of+bacterial+colony+patterns.">The significances of bacterial colony patterns.</a> Bioessays. 17, 597-597 (1995).</li><li>Shapiro, J., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinking+about+bacterial+populations+as+multicellular+organisms.">Thinking about bacterial populations as multicellular organisms.</a> Annu Rev Microbiol. 52, 81-81 (1998).</li><li>Engelberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+stalk-like+structures+in+Saccharomyces+cerevisiae.">Multicellular stalk-like structures in Saccharomyces cerevisiae.</a> J Bacteriol. 180, 3992-3992 (1998).</li><li>Scherz, R., Shinder, V., Engelberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+analysis+of+Saccharomyces+cerevisiae+stalk-like+structures+reveals+spatial+organization+and+cell+specialization.">Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization.</a> J Bacteriol. 183, 5402-5402 (2001).</li><li>Piccirillo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rim101p/PacC+pathway+and+alkaline+pH+regulate+pattern+formation+in+yeast+colonies.">The Rim101p/PacC pathway and alkaline pH regulate pattern formation in yeast colonies.</a> Genetics. 184, 707-707 (2010).</li><li>Sniegowski, P. D., Dombrowski, P. G., Fingerman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+and+Saccharomyces+paradoxus+coexist+in+a+natural+woodland+site+in+North+America+and+display+different+levels+of+reproductive+isolation+from+European+conspecifics.">Saccharomyces cerevisiae and Saccharomyces paradoxus coexist in a natural woodland site in North America and display different levels of reproductive isolation from European conspecifics.</a> FEMS Yeast Res. 1, 299-299 (2002).</li><li>Piccirillo, S., Honigberg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation+patterning+and+invasive+growth+in+wild+and+domesticated+yeast+colonies.">Sporulation patterning and invasive growth in wild and domesticated yeast colonies.</a> Res Microbiol. 161, 390-390 (2002).</li><li>Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+developing+multicellular+yeast+colony:+spatio-temporal+expression+of+Ato1p+ammonium+exporter.">Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter.</a> Environ Microbiol. , (2009).</li></ol>

Sunulan yöntem kolonilerin iç yapıları ortaya koymaktadır. S. bir dizi hücre tiplerinin kalıplarını belirleyen yöntem etkili olduğundan cerevisiae suşlarının farklı koloni morfolojileri ve aynı zamanda ilgili bir tür S. paradoksus 5, yöntem aynı zamanda geniş bir mantar ve diğer mikroorganizmalar üzerinde çalışmak için muhtemeldir . Yöntemin başarısı için kritik bir adım koloninin dahil olmak üzere tüm koloni, protokol boyunca ag...

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<td>Osmium tetroxide</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 19152</td>
<td>&nbsp;</td>
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<td>Silicone embedding molds</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>NC 9975029</td>
<td>&nbsp;</td>
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<td>Cycloaliphatic epoxide resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 15004</td>
<td>ERL 4221</td>
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<td>Epoxy resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 13000</td>
<td>DER 736</td>
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<td>Nonenyl Succinic Anhydride</td>
<td>&nbsp;</td>
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yeast colony embedding method

Embriyolar farklı hücre tipleri Desenlendirme metazoan gelişiminde kilit bir mekanizma. Koloniler ve biyofilm olarak mikroorganizmalar, topluluklar da hücre tiplerinin kalıplarını gösterilecek. Örneğin, maya S. cerevisiae, sporulated hücreleri ve pseudohyphal hücrelerin düzgün koloniler halinde dağıtılan değildir. Desenlendirme ve bu kalıplar altında yatan moleküler mekanizmaları fonksiyonel önemi halen yeterince anlaşılamamıştır. Mantar kolonilerin hücre tiplerinin kalıplarını soruşturma açısından bir sorun metazoan doku aksine, koloniler halinde hücrelerin nispeten zayıf birbirine bağlı olduğunu. Özellikle, mantar kolonileri, en dokularda bulunan ekstrasellüler matriks aynı geniş düzeyde içermez. İşte bu kolonilerin hücre tiplerinin iç desenler ortaya koymaktadır maya kolonileri gömme ve kesit için bir yöntem rapor. Bu yöntem, ışık mikroskobu ve transmisyon elektron mikroskopi için uygun ince bölümler (0,1 μ) için yararlıdır (0,5 μ) kalınlığında kesitler hazırlamak için kullanılabilir. Aşçı ve pseudohyphal hücreleri kolayca bu hücrelerin iç yapısı EM tarafından görüntülenmiştir olabilir, ışık mikroskobu ile oval maya hücreleri ayırt edilebilir. Yöntemi, agar kolonileri çevreleyen, onları Spurr orta infiltre ve sonra kesit dayanmaktadır. Çapı 1-2 mm aralığında bir Kolonileri, bu protokol için uygundur. Kolonilerin iç görüntülenmesine ek olarak, yöntem temel agar işgal koloni bölgesi görselleştirme sağlar.

Watch this Scientific Journal Video about Yeast Colony Embedding Method at JoVE.com

Yeast Colony Embedding Method

Işık ve elektron mikroskopi için kesit maya kolonileri izin gömmek için bir yöntem. Bu protokol, bir mantar toplum içinde hücre tiplerinin organizasyon anlayışına yönelik yeni bir araç sağlayan koloniler içinde sporulated hücreleri ve pseudohyphal hücrelerin dağılımı belirliyor.

Maya Colony Gömme Yöntemi

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Research

JoVE Journal

Biology

11.4K Görüntüleme.  University of Missouri - Kansas City. Işık ve elektron mikroskopi için kesit maya kolonileri izin gömmek için bir yöntem. Bu protokol, bir mantar toplum içinde hücre tiplerinin organizasyon anlayışına yönelik yeni bir araç sağlayan koloniler içinde sporulated hücreleri ve pseudohyphal hücrelerin dağılımı belirliyor.

Video: Yeast Colony Embedding Method

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Plastik gömülmesi ve Xenopus laevis Embriyolar Kesit

Biyoloji

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Geliştirici Maya replikatif Ömrü Ölçüm

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Maya hücreleri Mitokondri saflaştırılması

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video Biyoinformatik İnsan Embriyonik Kök Hücre Colony Büyüme Analizi

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

Yüksek verim Maya Plazmid overekspresyonu Ekran

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Barkodlu Maya Kütüphaneler Rekabet Genomik Ekranlar

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

İnsülin ifade Koloni oluşturan atalarıdır için Kantitatif Testi

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Yumuşak Agar koloni formasyon deneyi

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

Bölünme Maya Cinsel Yaşam Döngüsü Mikroskopi

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY: Maya Büyümesinin Çok Parametreli Niceleşmesi İçin Büyük Ölçekli Bir Yöntem

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...