Name: flow cytometry-based purification of s. cerevisiae zygotes
Uploaded: 2012-09-21T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry-based Purification of S. cerevisiae Zygotes at JoVE.com

Vi takker Dr. R. Michael Sramkoski for hjælp med flowcytometri, Dr. Purnima Jaiswal og Dr. Di Wu for tidligere input og Ilya Aylyarov om hjælp med illustrationerne. Støttet af NIH Grant R01GM089872 og Cytometry &amp; Imaging Microscopy Core Facility for Case Comprehensive Cancer Center (P30 CA43703).

1. Haploide Cell Growth <ol><li> Grow S. cerevisiae-celler fra S288C baggrund (BY4741) under standardbetingelser 10. </li><li> Omdan haploider at udtrykke en af to iøjnefaldende fluorescerende markører 10. De markører er opløselige GFP udtrykkes i cytoplasmaet (pEG220, URA3/YIp, lineariseret med BglII 11) og en enkelt ribosomale protein, RPL25, fusioneret med mCherry (pAJ1661, URA3/CEN, fra A. Johnston). Vi forventer, at ækvivalente tofarvet oprensningsprotokoller kunne g...

<ol>
	<li>Herskowitz, I., Oshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Control of cell type in Saccharomyces cerevisiae: Mating type and mating-type interconversion. , 181-210 (1981).</li><li>Muller, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parental+age+and+the+life-span+of+zygotes+of+Saccharomyces+cerevisiae.">Parental age and the life-span of zygotes of Saccharomyces cerevisiae.</a> Antonie Van Leeuwenhoek. 51, 1-10 (1985).</li><li>Tartakoff, A. M., Jaiswal, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+fusion+and+genome+encounter+during+yeast+zygote+formation.">Nuclear fusion and genome encounter during yeast zygote formation.</a> Mol. Biol. Cell. 20, 2932-2942 (2009).</li><li>Azpiroz, R., Butow, 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=Patterns+of+mitochondrial+sorting+in+yeast+zygotes.">Patterns of mitochondrial sorting in yeast zygotes.</a> Mol. Biol. Cell. 4, 21-36 (1993).</li><li>Rose, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+fusion+in+the+yeast+Saccharomyces+cerevisiae.">Nuclear fusion in the yeast Saccharomyces cerevisiae.</a> Annu. Rev. Cell Dev. Biol. 12, 663-695 (1996).</li><li>Dujon, B., Jones, E., Strathern, J., Broach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance. , 505-635 (1981).</li><li>Sprague, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+yeast+mating+reaction.">Assay of yeast mating reaction.</a> Methods Enzymol. 194, 77-93 (1991).</li><li>Ydenberg, C. A., Rose, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mating:+a+model+system+for+studying+cell+and+nuclear+fusion.">Yeast mating: a model system for studying cell and nuclear fusion.</a> Methods Mol. Biol. 475, 3-20 (2008).</li><li>Sena, E. P., Radin, D. N., Fogel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronous+mating+in+yeast.">Synchronous mating in yeast.</a> Proc. Natl. Acad. Sci. U.S.A. 70, 1373-1377 (1973).</li><li>Amberg, D., Burke, D., Strathern, D. <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 Edition. , (2005).</li><li>Nolan, S., Cowan, A. E., Koppel, D. E., Jin, H., Grote, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FUS1+regulates+the+opening+and+expansion+of+fusion+pores+between+mating+yeast.">FUS1 regulates the opening and expansion of fusion pores between mating yeast.</a> Mol. Biol. Cell. 17, 2439-2450 (2006).</li><li>Weisman, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelles+on+the+move:+insights+from+yeast+vacuole+inheritance.">Organelles on the move: insights from yeast vacuole inheritance.</a> Nat. Rev. Mol. Cell Biol. 7, 243-252 (2006).</li></ol>

Tilgængeligheden af ​​oprensede zygoter bør lette biokemiske undersøgelser, herunder transcriptomics, proteomics og lipidomics samt undersøgelse af mekanismer, hvorved zygoter undergår cellecyklus genindrejse, cellevæg remodeling, spirende og arv egenskaber, etc. Alternativt kunne zygoter blive genereret start med stammer, der bærer karakteristiske mutationer i en eller begge forældre. Endvidere er de oprensede zygoter, som for haploide eller diploide celler, kan fryses i medium indeholdende 15% glycerol og ...

<table border="1"><tbody><tr><td> Reagens / Udstyr </td><td> Kilde </td><td> Katalognummer </td></tr><tr><td> pEG220, URA3/YIp, der koder cytoplasmatiske GFP 11) </td><td> E. Grote </td><td></td></tr><tr><td> pAJ1661, URA3/CEN, der koder RPL25 fusioneret til mCherry </td><td> A. Johnston </td><td></td></tr><tr><td> Fisher FB120 sonikator </td><td> Fisher Scientific </td><td></td></tr><tr><td> Polypropylenrør med filter cap </td><td> BD Falcon </td><td> Ref 352.235 </td></tr><tr><td> Afkølet mikrocentrifuge </td><td> Tomy </td><td> MRX-150 </td></tr><tr><td> Flowcytometri kappe opløsning </td><td> Biosure </td><td> 1027 </td></tr><tr><td> iCyt Reflection Model Flow Cytometer </td><td> Sony </td><td></td></tr><tr><td> DeltaVision dekonvolution mikroskop </td><td> Anvendt Precision </td><td></td></tr><tr><td> Opretstående epifluorescensmikroskop </td><td> Leica </td><td></td></tr><tr><td> UltraPure Agarose </td><td> Invitrogen </td><td> 15.510-07 </td></tr><tr><td> CSM glucose formulering Bemærkninger: For agarplader, nøjes med 2% agar (Difco) </td><td> 10 </td><td></td></tr></tbody></table>

flow cytometry-based purification of s. cerevisiae zygotes

Zygoter er vigtige mellemprodukter mellem haploide og diploide stater i livscyklus mange organismer, herunder gær (figur 1) 1. S. cerevisiae zygoter resultat af fusion af haploide celler af forskellig parringstype (MATa, MATalpha) og give anledning til tilsvarende stabile diploider, der successivt genererer så mange som 20 diploid afkom som følge af deres påfaldende asymmetriske mitotiske afsnit 2. Zygote formation organiseret af en kompleks sekvens af begivenheder: I denne proces binder opløselige hinanden passende faktorer til beslægtede receptorer, udløser receptormedierede signaleringskaskader, der letter afbrydelse af cellecyklussen og de samles i celle-cellefusion. Zygoter kan betragtes som en model for stamcelle eller stamcelle-funktion. Selv om meget er blevet lært om dannelsen af ​​zygoter og selvom zygoter er blevet anvendt til at undersøge celle-molekylære spørgsmål af generel betyicance, næsten alle studier har gjort brug af samvirkende blandinger, hvori zygoter er blandet med et flertal population af haploide celler 3-8. Mange aspekter af biokemi af zygote dannelse og den fortsatte liv i det befrugtede æg derfor fortsat ikke undersøgt. Rapporter om oprensning af gær zygoter beskriver protokoller baseret på deres sedimentation egenskaber 9; dog dette sedimentation-baserede procedure ikke giver næsten 90% renhed i vores hænder. Desuden har den den ulempe, at celler udsættes for hypertonic sorbitol. Vi har derfor udviklet en alsidig oprensningsprocedure. Til dette formål blev par af haploide celler, der udtrykker røde eller grønne fluorescerende proteiner co-inkuberes for at tillade zygote formation, høstet på forskellige tidspunkter, og de resulterende zygoter blev oprenset under anvendelse af en flow-cytometri-baseret sortering protokol. Denne teknik giver en bekvem visuel vurdering af renhed og modning. Den gennemsnitlige renhedsgradaf fraktionen er omkring 90%. Ifølge timingen af ​​høsten, kan zygoter af varierende grader af modenhed skal tilbagebetales. De oprensede prøver giver et bekvemt udgangspunkt for &quot;-miske&quot; undersøgelser, til inddrivelse af initial afkom, og for systematisk undersøgelse af denne progenitorcelle.

Watch this Scientific Journal Video about Flow Cytometry-based Purification of S. cerevisiae Zygotes at JoVE.com

Flow Cytometry-based Purification of S. cerevisiae Zygotes

For at oprense zygoter af<em&gt; S. cerevisiae</em&gt;, Blev haploide celler af modsat parringstype manipuleret til at udtrykke rødt eller grønt fluorescerende proteiner, co-inkuberede at tillade zygote formation og fraktioneret under anvendelse af en flow-cytometri-baseret protokol. Det stærkt fraktion muliggør efterfølgende &quot;-miske&quot; undersøgelser, inddrivelse af initial afkom, og systematisk undersøgelse af zygote morfogenese.

Flowcytometri-baseret oprensning af<em&gt; S. cerevisiae</em&gt; Zygoter

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

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Biology

Flow Cytometry-based Purification of S. cerevisiae Zygotes

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, ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

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

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 ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

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Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Ekspression og oprensning af cystisk fibrose-transmembrankonduktans-regulator protein i Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Optimeret farvning og spredning modelleringsmetoder for celledeling Overvågning ved hjælp cellesporing Farvestoffer

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

High Throughput Microinjections of Sea Urchin Zygotes

High Throughput Microinjections af søpindsvin Zygoter

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...