Yazarlar Matthias Peter ve bu yöntemler geliştirilmiştir Zürich ETH Biyokimya Enstitüsü&#39;nde yaptığı grup ederim. Bu çalışma, İsviçre Ulusal Bilim Vakfı tarafından desteklenmiştir.

1.. Mikroskopi Ölçümler <ol><li> Maya sentetik ortam 5 ml inoküle. </li><li> Gece boyunca 30 ° C&#39;de hücreler büyür. </li><li> Ertesi sabah, gece boyunca kültürün OD 600 ölçün. </li><li> 600 0.05 OD 5 ml sentetik kültür ortamı içinde bir gece boyunca seyreltin. </li><li> En az 4 saat boyunca 30 ° C&#39;de seyreltilmiş kültür büyütün. </li><li> Hazırlama 3 kat sentetik aracı madde içinde (0.6 M) NaCl solüsyonu konsantre edildi. </li><li> PBS, Concanavalin A (ConA) bir 1 mg / ml çözelti hazırlayın. </li><li> Süzüntü ~ iyi slayt içine ConA&#39;nın çözeltisi 200 ul. </li><li> 30 dakika bekletin. </li><li> ConA&#39;nın çözüm çıkarın. </li><li> De 150 ul H 2 O ekleyin. </li><li> H 2 O. kaldır </li><li> Hücre kültürü OD 600 ölçün. </li><li> 1.5 ml = 0.02 mikrotüp içinde OD 600 hücre kültürünün 300 ul hazırlayın. </li><li> 45 saniye için bir su banyosu içinde hücreleri sonikasyon. </li><li> Vortex kısaca microtube. </li><li> 45 saniye için bir su banyosu içinde hücreleri sonikasyon. </li><li> Kısaca Vortex Mikrotüp. </li><li> Oyuğuna hücre kültürünün 200 ul ekleyin. </li><li> Hücreler kuyunun dibine çöker izin 30 dakika bekleyin. </li><li> Mikroskop iyi slayt yerleştirin. </li><li> Kaydedilecek floresan kanal aydınlatma ayarlarını seçin. </li><li> (: Hücrelerinin uygun bir segmentasyon sağlamak için odak düzleminin altında 3 um biraz odak dışında biri) iki parlak bir alan görüntüler için aydınlatma ayarlarını seçin. </li><li> Time-lapse ölçümü için zaman aralıklarını seçin </li><li> Görüntü görüş alanları seçin. </li><li> Bir kaç kare alımı başlatabilirsiniz. </li><li> 0.6 M NaCl ortamında 100 ul eklemek için satın Pause. </li><li> Görüntüleme Devam. </li></ol> 2. Sitometrisi Ölçümler <ol><li> Maya sentetik ortam 5 ml inoküle. </li><li> Gece boyunca 30 ° C&#39;de büyür. </li><li> TedbirOD, gece boyunca kültürün 600 ertesi sabah. </li><li> 600 0.1 OD 5 ml sentetik bir ortam içinde bir gece boyunca kültür seyreltin. </li><li> OD 0,2-0,4 600 ulaşmak için en az 4 saat boyunca 30 ° C&#39;de büyür. </li><li> H2O içinde sikloheksimid çözelti 1 mg / ml hazırlanması </li><li> Hazırlama 3 kat sentetik aracı madde içinde (0.6 M) NaCl solüsyonu konsantre edildi. </li><li> Ölçülecek her zaman noktası için 0.6 M NaCl ortamın 100 ul bir 1.5 ml mikrotüp hazırlayın. </li><li> 0 zamanında, her bir mikrotüp hücre kültürü 200 ul ekleyin. </li><li> 30 ° C&#39;de çalkalayarak kuluçkalayın </li><li> T süresi de, bir mikrotüpe 30 ul sikloheksimid ekleyin. </li><li> Her istenilen zaman noktaları için adımı 2.11 tekrarlayın. </li><li> En az 45 dakika boyunca tüm mikrotüpler inkübe edin ve çalkalanarak 30 ° C&#39;de 3 saat kadar. </li><li> 400 ul PBS ile FACS tüpleri hazırlayın. </li><li> 1 dakika süre ile bir su banyosunda sonikasyon hücreleri. </li><li> Kısaca Vortex mikrotüpler. </li><li> Oğlum1 dakika boyunca bir su banyosu içinde hücreleri TARĐHĐ BELGE. </li><li> Kısaca Vortex mikrotüpler. </li><li> Her mikrotüpünden olarak karşılık gelen FACS tüpüne 100 ul hücre kültürü ekleyin. </li><li> Tespiti için 488 nm uyarma lazer ve 530/30 nm band geçiren filtre seçin. </li><li> Testi olmayan ifade ve tam onlar dedektör hassasiyet aralığında düşüş olmadığını doğrulamak için örnek ifade. </li><li> Alınan her bir örnek için 10,000 hücre ölçün. </li></ol>

<ol>
	<li>Gasch, A. P., Spellman, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+expression+programs+in+the+response+of+yeast+cells+to+environmental+changes.">Genomic expression programs in the response of yeast cells to environmental changes.</a> Molecular biology of the cell. 11 (12), 4241-4257 (2000).</li><li>de Nadal, E., Zapater, M., Alepuz, P. M., Sumoy, L., Mas, G., Posas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MAPK+Hog1+recruits+Rpd3+histone+deacetylase+to+activate+osmoresponsive+genes.">The MAPK Hog1 recruits Rpd3 histone deacetylase to activate osmoresponsive genes.</a> Nature. 427 (6972), 370-374 (2004).</li><li>Ghaemmaghami, S., Huh, W. -. 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B., Levine, A. J., Siggia, E. D., Swain, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+gene+expression+in+a+single+cell.">Stochastic gene expression in a single cell.</a> Science. 297 (5584), 1183-1186 (2002).</li><li>Pelet, S., Dechant, R., Lee, S. S., van Drogen, F., Peter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+image+analysis+platform+to+quantify+signal+transduction+in+single+cells.">An integrated image analysis platform to quantify signal transduction in single cells.</a> Integrative biology : quantitative biosciences from nano to macro. 4 (10), 1274-1282 (2012).</li><li>Carpenter, A. E., Jones, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome biology. 7 (10), R100 (2006).</li><li>Chernomoretz, A., Bush, A., Yu, R., Gordon, A., Colman-Lerner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Cell-ID+1.4+with+R+for+microscope-based+cytometry.">Using Cell-ID 1.4 with R for microscope-based cytometry.</a> Current protocols in molecular biology. Chapter 14, Unit 14.18 (2008).</li><li>Dimopoulos, S., Mayer, C., Rudolf, F., Stelling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+Single+Cell+Segmentation+in+a+Variety+of+Microscopy+Images.">Automatic Single Cell Segmentation in a Variety of Microscopy Images.</a> Current protocols in molecular biology. , (2012).</li><li>Miyawaki, A., Nagai, T., Mizuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+protein+fluorophore+formation+and+engineering.">Mechanisms of protein fluorophore formation and engineering.</a> Current Opinion in Chemical Biology. 7 (5), 557-562 (2003).</li><li>Hersen, P., McClean, M. N., Mahadevan, L., Ramanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+processing+by+the+HOG+MAP+kinase+pathway.">Signal processing by the HOG MAP kinase pathway.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (20), 7165-7170 (2008).</li><li>Pepperkok, R., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+fluorescence+microscopy+for+systems+biology.">High-throughput fluorescence microscopy for systems biology.</a> Nature reviews Molecular cell biology. 7 (9), 690-696 (2006).</li></ol>

Yazarlar, hiçbir rakip mali çıkarlarını olmadığını beyan ederim.

Mikroskopla inceleme ConA ile kuyunun tedavisi, hücrelerin uygun bir görüntüleme sağlamak için önemli bir adımdır. ConA PBS (5 mg / mi) içinde düşük bir çözünürlüğe sahip olduğundan, filtreleme işlemi sağlar: filtre çözelti içinde mevcut olan ve görüntüleme müdahale büyük agregaların çıkarılması. Hücreler, işlemden geçirilen yüzeye bağlı olarak güçlü bir şekilde tutunur ve uyaran çözeltisinin eklenmesinin uyaran önce ve sonra, bir sürekli izleme...

Sinyal transdüksiyon cascades ile genellikle proteinlerin ekspresyonu son bulur. Bu ifade profilinin karakterizasyonu biyolojik yollarının işlevini anlamada önemli bir unsurdur. Up-regüle proteinler ve bunların aktivasyon dinamikleri spektrumunun tanımlanması, bu tür mikro-dizileri, kuzey ya da batı lekeleri blotlar 1-3 gibi çeşitli teknikler ile elde edilebilir. Bununla birlikte, bu teknikler arasında, hücreler bütün bir nüfusun ortalama tepki. Protein ekspresyonunun ince düzenleme anlamak için, tek hücre düzeyinde ölçümleri toplamak için tercih edilir. İdeal olarak, bu ölçümler de altta yatan yolun matematiksel modeller geliştirmek için uygun nicel veri sağlamalıdır. Mikroskopi ve akış sitometri ideal gibi nicel tek hücre ölçümlerini sunmak için uygundur iki teknik vardır. Bir floresan protein ile proteinlerin endojen etiketleme be ekspresyon seviyesi 4 ölçmek için kullanılır. Proteinin terminalinde büyük bir floresan yarımının eklenmesi işlevsel olmayan işlemek için Bununla birlikte, bu bir floresan yapısının ekspresyonunu tahrik eden bir promotere göre belirli ifade muhabir oluşturmak için genellikle daha fazla tercih edilir. Bu, raportör protein hücresel sistem için dışsal ve bu nedenle hücre içinde yer alıyor sinyal olaylarını etkilememektedir. Mikroskopi ve akış sitometrisi her ikisi yaygın olarak maya sinyal alanında kullanılmıştır. Örnek olarak, Colman-Lerner ve arkadaşları korelasyon, tek hücreler içinde, maya eşleşme sinyal iletim 5 gürültüyü ölçmek için mikroskopi ile spesifik ve yapısal olarak ifade edilen muhabir çiftleşme ifade Acar ve düzenleyici ağ incelemek için akış sitometrisi el arkadaşları GAL genlerin 6 ekspresyonunu kontrol etmektedir. Önceki bir çalışmada 7&#39;de, bir combinati kullandıkozmotik basıncı yüksek gliserol maya tomurcuklanma olarak (HOG) yolunun gelen ifade çıkışını incelemek için bu iki teknikleri üzerine. Bu, mitojen tarafından aktive edilen protein kinaz (MAPK) yolu hiper-osmotik stres tarafından tetiklenir. Bu, yaklaşık 300 genlerin ekspresyonu ile sonuçlanan bir transkripsiyon programı indükleme hücrenin çekirdeğine doğru Hog1 MAPK aktivasyonu ile sonuçlanır. Bu işlemi incelemek için, bir dört Venus floresan proteini (p-STL1 QV) ekspresyonunu tahrik STL1 promoteri (Hog1 aktivitesi 8&#39;e yanıt olarak, özellikle kaynaklı bir gen) dayanan bir sentezleme raportör mühendisliği vardı. Ölçümler, flüoresan raportör ifade nüfusun sadece bir kısmı ile orta gerilim seviyesinde (0.1 M NaCl) ile iki hücre popülasyonlarının varlığını ortaya sitometrisi akış. Biz daha bu davranışı araştırmak için mikroskobu kullandılar ve protein ifadesindeki bu gürültü içsel faktörlere 9 tabi olduğunu keşfetti. Biz could ayrıca Hog1 aktivite benzer bir düzeyde olan hücreler çarpıcı farklı ifade sonuçlarını görüntülemek verebilecek görüyoruz. Bu iki tekniğin kombinasyonu bize stres yanıtı genlerinin yavaş yeniden tek hücre düzeyinde 7 de ifade sonucunu nasıl etkilediğini göstermek için izin verdi. Bu yazıda, mikroskobu ile protein ifade miktarının bir örnek olarak hiper-ozmotik şok tarafından uyarılan p STL1-QV muhabiri ifadesini kullanın ve akım sitometri. 0.2 M NaCl strese maruz aynı gerilme hem tekniklerle incelenmiştir. Bu bize bu iki derece tamamlayıcı teknikler arasındaki bazı temel farklılıkları vurgulamak için izin verecektir.

<table border="1">
		<tbody>
		 <tr>
			 <td></td>
			 <td></td>
			 <td></td>
			 <td>Reagent</td>
		 </tr>
		 <tr>
			 <td>Yeast Nitrogen Base</td>
			 <td>ForMedium</td>
			 <td>CYN6202</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>CSM amino-acid mix</td>
			 <td>ForMedium</td>
			 <td>DCS0011</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Concanavalin A</td>
			 <td>GE Healthcare</td>
			 <td>17-0450-01</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Cycloheximide</td>
			 <td>Fluka Aldrich Sigma</td>
			 <td>C7698</td>
			 <td>Toxic</td>
		 </tr>
		 <tr>
			 <td>ySP9</td>
			 <td></td>
			 <td></td>
			 <td>W303Mata leu2::LEU2-pSTL1-quadrupleV enus</td>
		 </tr>
		 <tr>
			 <td>pSP34</td>
			 <td></td>
			 <td></td>
			 <td>pRS305 pSTL1 (-800 -0) - quadruple V enus</td>
		 </tr>
		 <tr>
			 <td></td>
			 <td></td>
			 <td></td>
			 <td>Material</td>
		 </tr>
		 <tr>
			 <td>Microscope</td>
			 <td>Nikon</td>
			 <td>Ti-Eclipse</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Microscope control software</td>
			 <td>Micro-manager</td>
			 <td>Ver 1.4.11</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Incubation chamber</td>
			 <td>LIS</td>
			 <td>The Box</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Fluorescence light source</td>
			 <td>Lumencor</td>
			 <td>SpectraX</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Camera</td>
			 <td>Hamamatsu</td>
			 <td>Flash 4.0</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Flow cytometer</td>
			 <td>BD</td>
			 <td>FACS calibur</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>Sonicator bath</td>
			 <td>Telesonic</td>
			 <td>TUC-150</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>8-well-slide </td>
			 <td>Thermo Lab-tek</td>
			 <td>155409</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>96-well-plate</td>
			 <td>Matrical bioscience</td>
			 <td>MGB096-1-2-LG</td>
			 <td></td>
		 </tr>
		 <tr>
			 <td>FACS tube</td>
			 <td>BD Falcon</td>
			 <td>352054</td>
			 <td></td>
		 </tr>
		</tbody>
 </table>

temporal quantification of mapk induced expression in single yeast cells

Tek hücre düzeyinde gen ifadesinin miktar nüfus düzeyinde gerçekleştirilen ölçümler gölgede yeni düzenleme mekanizmalarını ortaya çıkarır. İki yöntem mikroskopi dayalı ve akım sitometri gibi veriler elde edilebilir nasıl göstermek için sunulmaktadır. Mayada, ozmotik basıncı yüksek gliserol MAPK yolağının aktivasyonu ile uyarılan bir flüoresan raportör ekspresyonu, bir örnek olarak kullanılmıştır. Her bir yöntemin özel avantajları vurgulanır. Sitometrisi hücrelerin geniş bir sayısı (10,000) büyüklüğüne ve floresan proteinin yavaş olgunlaştırma kinetik bağımsız protein ifade dinamikleri doğrudan bir ölçümünü sağlar akış. Mikroskobu ile canlı hücrelerin Imaging az hücrelerinde raportör olgunlaşmış formunun ölçümü ile sınırlı aksine gereğidir. Ancak, bu teknikle üretilen veri setleri birden gazetecilere kombinasyonları ve mekansal ve zamansal bilgiler son derece zengin sayesinde olabilirtek tek hücrelerden elde. Bu iki ölçüm yöntemlerinin kombinasyonu sinyal yolları ile protein ifadesinin düzenlenmesinde yeni anlayışlar sunabilirsiniz.

Mikroskopla inceleme Sentezleme raportör p STL1-QV (ySP9 7) taşıyan maya hücreleri iyi slaydın tabanına bağlanmış ve mikroskop altında yerleştirildi. Hücreler görüntüleme oturumu sırasında doğrudan kuyuya stres ortamının eklenmesi ile uyarıldı. Bu bize yol indüksiyon öncesi hücrelerin birkaç görüntü elde ve stimülasyon sonrasında kendi kaderini takip sağlar. Bu durumda, hücreler, 10 dakika bir zaman aralığı ile ~ 2 saat boyunca takip edild...

Watch this Scientific Journal Video about Temporal Quantification of MAPK Induced Expression in Single Yeast Cells at JoVE.com

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

Akış sitometrisi ve mikroskopiye dayanan iki yardımcı yöntem mayada bir MAPK yolağının aktivasyonu ile uyarılan gen ekspresyonunun dinamiklerinin tek hücre düzeyinde miktarının, ki olanak sunulmaktadır.

Tek Maya Hücreleri MAPK İsteyerek İfade zamansal Kantitasyonu

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

temporal-quantification-mapk-induced-expression-single-yeast

Research

JoVE Journal

Biology

8.4K Görüntüleme.  University of Lausanne.  Akış sitometrisi ve mikroskopiye dayanan iki yardımcı yöntem mayada bir MAPK yolağının aktivasyonu ile uyarılan gen ekspresyonunun dinamiklerinin tek hücre düzeyinde miktarının, ki olanak sunulmaktadır. 

Video: Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 1 of 3.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: Bölüm 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

Biyoloji

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: Part 2

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: Bölüm 3

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Rekombinant Proteinlerin Metilotropik maya İfade Pichia pastoris</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Büyüme inhibisyonu fenotipleri tanımlanması Mayalarda bakteriyel Tip III Efektör İfade İndüklenmiş

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Gen İfadesi Çalışmaları basitleştirin Otomatik Hücre Sayıcı kullanma: Namalwa Hücreler IL-4 Bağımlı Gen İfadesi siRNA demonte

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Ününüz Aşağıdaki Tek Hücre Düzeyinde Küresel RNA Sentezi Analizi

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Tütün Geçici İfade Sisteminin Kullanılması bir MAPK sinyal yolu Mekansal Reorganizasyon Görüntüleme

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Mikroakışkan Aygıtlar Kullanma Tek Budding Maya Hücreleri ömrü ve Hücresel fenotipleri Ölçmek için

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...