We thank Sue Lindquist, Aaron Gitler, and Martin Duennwald for kindly sharing reagents. Our studies were supported by: an American Heart Association Post-Doctoral Fellowship (M.E.J); NIH Director's New Innovator Award DP2OD002177, NIH grants R21NS067354, R21HD074510, and R01GM099836, a Muscular Dystrophy Association Research Award (MDA277268), Packard Center for ALS Research at Johns Hopkins University, Target ALS, and an Ellison Medical Foundation New Scholar in Aging Award (J.S.).

1. Biblioteca Generation <ol><li> Per costruire librerie di Hsp104 utilizzando soggetto a errori PCR specifico del dominio, prima amplificare il dominio di interesse con un errore DNA polimerasi inclini 20. </li><li> Purificare il prodotto di PCR mediante estrazione gel. </li><li> Eseguire un passo estensione megaprimer utilizzando un protocollo standard di mutagenesi sito-diretta: unire 50 ng modello plasmide, 250 ng megaprimer, 200 mM dNTP, e ad alta fedeltà polimerasi DNA in tampone PCR, e portare a 50 ml volume totale di acqua di grado PCR 20 . Eseguire un programma standard di PCR. NOTA: primer specifici usati varieranno in base alla particolare regione del gene che deve essere amplificato. <ol><li> A seguito di PCR, digerire il DNA dei genitori modello con 1 ml Dpn I restriction enzyme per 2 ore a 37 ° C. </li></ol></li><li> Trasforma la libreria di elettroporazione o altri mezzi a ultracompetent E. coli e purificarlo da miniprep. NOTA: generazione Bibliotecavariabile in base sostanzialmente sugli obiettivi di un dato esperimento. Hsp104 è una grande proteina, quindi non è pratico di randomizzare l&#39;intero gene, che è per questo che utilizziamo errore specifico del dominio incline PCR. Inoltre, la struttura di Hsp104 rimane poco compresa, rendendo la progettazione di librerie diretti impegnativi 21. Le librerie possono essere costruiti utilizzando approcci dirette o casuali per mutagenesi, con l&#39;unica limitazione è che il plasmide ossatura modello deve contenere il gene URA3 per consentire 5-FOA contatore scelta su supporti destrosio. </li></ol> 2. Trasformazione della Biblioteca Hsp104 <ol><li> Integrare il substrato associata a malattia in lievito Hsp104 W303aΔ con un acetato di litio standard di / PEG protocollo di trasformazione 22. Selezionare singole colonie e lo screening per la tossicità per isolare un ceppo con elevata tossicità della malattia substrato 23 associati. NOTA: Utilizzare un ceppo integrato e isolaTé una singola colonia per garantire la parità di espressione in tutte le cellule. Clone substrato malattia in un plasmide permette l&#39;integrazione del gene sotto qualsiasi marcatore diverso uracile (usiamo istidina) e la libreria Hsp104 nel plasmide pAG416GAL (marcatore uracile) 24. Per consentire 5-FOA counterselection, assicurarsi che la libreria è espresso da un plasmide con il marcatore uracile. Altri ceppi di lievito possono anche essere impiegati. Abbiamo notato un simile fenotipo soppressione di tossicità utilizzando W303a e BY4741 ceppi di lievito in entrambi gli sfondi Hsp104 WT e Δ (MEJ e JS, osservazioni non pubblicate). </li><li> Trasforma la libreria Hsp104 in questo ceppo utilizzando lo stesso acetato di litio / PEG protocollo di trasformazione 22. Scale la trasformazione appropriato per mantenere la sequenza spazio della biblioteca e di preservare la dimensione biblioteca predetto. </li><li> Piatto il composto trasformazione su noninducing, piastre selettive (ad esempio, SD-His-Ura) con un numero sufficiente di piastre aassicurare la crescita di un gran numero di colonie. Utilizzare grandi piastre di Petri (150 mm) per ridurre al minimo il numero di piatti necessari. Recupero trasformanti che utilizzano piastre permette l&#39;efficienza di trasformazione da valutare. </li><li> Trasforma Hsp104 WT e controlli negativi vettoriali in parallelo. </li></ol> 3. Screening per la repressione del proteotoxicity <ol><li> Lavare le colonie al largo delle piastre con raffinosio integrato dei media di abbandono (ad esempio SRaff-His-Ura). Utilizzare una pipetta sierologica e applicatori in legno sterili per allentare le colonie dalle piastre. Trasferire i lavaggi liquido in una provetta da 50 ml e agitare accuratamente per separare eventuali grumi di cellule. Diluire a una cultura un po &#39;nuvoloso, OD 600 ~ 0.025. NOTA: Qui serve raffinosio per alleviare le cellule della repressione di glucosio-mediata di induzione, adescamento in tal modo le cellule per l&#39;induzione galattosio-mediata. </li><li> Far crescere la coltura diluita durante la notte nei mezzi di abbandono raffinosio con agitazione a 30 ° C. Far crescere la Hsp104 WT e controlli vettoriali in parallelo. </li><li> La mattina seguente, piatto una serie di concentrazioni su galattosio individuale (ad esempio SGal-His-Ura) piastre (1 ml a 2 ml cultura concentrate in 400 ml di volume totale). Placcatura una vasta gamma di concentrazioni garantirà che un piatto avrà singole colonie. La concentrazione effettiva richiesta dipende dalla tossicità del substrato patologia di interesse. </li><li> Normalizza il vettore e culture Hsp104 WT al diametro esterno 600 della biblioteca e piastra volumi uguali di confrontare la crescita della biblioteca rispetto ai controlli. </li><li> Per valutare selezione rigore, piatto biblioteca in glucosio (repressione) dei media. Incubare le piastre per 2-3 giorni a 30 ° C, fino a quando le colonie appaiono (Figura 2). Valutare le colonie risultanti e confrontare ai controlli. NOTA: Spesso entrambe le colonie grandi e piccole si ottengono, ma nessuna correlazione tra le dimensioni delle colonie e di agireivity si osserva. Schermo questi potenziali colpi utilizzando una tecnica di selezione contatore 5-FOA (Fase 4) per ridurre al minimo i falsi positivi riportati al sequenziamento. </li></ol> 4. 5-FOA Counterselection e Spotting per eliminare i falsi positivi <ol><li> Streak le singole colonie in duplice copia su supporto matrimoniale e letto singolo caduta di tensione (ad esempio, SD-suo-Ura e SD-I suoi piatti) e crescere una notte a 30 ° C. Ripetere con controlli vettoriali e Hsp104 WT. Placcatura su SD-His prima 5-FOA aumenta l&#39;efficienza della fase 5-FOA aumentando la probabilità che le cellule avranno perso il plasmide Hsp104 quando vengono piastrate su 5-FOA. </li><li> Per evitare che le piastre si secchi, avvolgere le piastre in parafilm suoi Ura-SD-e conservare a 4 ° C durante l&#39;esecuzione dello schermo 5-FOA. Queste piastre saranno eventualmente utilizzati per il sequenziamento. </li><li> Streak le colonie dalla SD-I suoi piatti a singole colonie su piastre 5-FOA (YPD supporti + 1 g / L 5-FOA) e incubare per 1-2 giorni a 30 &amp;# 186; C fino singole colonie appaiono. Qui, 5-FOA viene convertito in un prodotto tossico (5-fluorodeossiuridina) in cellule che contengono il gene URA3. Pertanto, eventuali cellule ancora ospitano il plasmide Hsp104 moriranno. </li><li> Streak le singole colonie dalle piastre 5-FOA in duplicato su SD-Ura e SD-I suoi piatti. Test 3 colonie per ogni hit per aumentare la probabilità che almeno una colonia per ogni colpo avrà perso il plasmide Hsp104. Incubare per 1-2 giorni a 30 ° C. Le colonie che hanno perso il plasmide Hsp104 si svilupperà su SD-I suoi piatti, ma non piatti SD-Ura. </li><li> Far crescere le tensioni che hanno perso i plasmidi Hsp104 per individuare saggi. Crescere i ceppi di saturazione in raffinosio abbandono dei media (ad esempio SRaff-His) in 96 Deepwell 2 piastre ml notte a 30 ° C con agitazione. Assicurati di includere i ceppi di controllo 5-FOA trattati. </li><li> Preparare piastre per individuare test. Utilizzando una pipetta multicanale, un&#39;aliquota raffinosio 200 microlitri mezzi di abbandono (ad esempio SRaff-His) perpozzetto di una piastra da 96 pozzetti, riservando colonne 1 e 7 per le culture pulito. Aliquotare 250 ml di ciascuno dei 16 culture saturi nelle colonne 1 e 7 della piastra. </li><li> Serialmente diluire le culture 5 volte in ogni colonna della piastra, mescolando accuratamente con una pipetta multicanale. Rimuovere 50 ml di coltura diluita dalle colonne 6 e 12 per garantire il volume finale di ciascun pozzetto a 200 microlitri. Questo è essenziale per garantire anche spotting. </li><li> Utilizzando uno strumento replicatore 96-bullone (Frogger), individuare le culture in duplicato su SD-Sue e-SGal suoi piatti. Incubare le piastre a 30 ° C per 2-3 giorni. </li><li> Seleziona per sequenziamento ceppi che presentano tossicità sui SGal-sue piastre simile a quella dei controlli. Eliminare positivi come falsi ceppi che non sono tossici come i controlli. 5-FOA è anche spesso tossici propria, così scartare ceppi che presentano un difetto di crescita su SD-sue piastre o che mostrano sostanzialmente maggiore tossicità rispetto al substrato malattie sola ( <strong&gt; Figura 3). </li></ol> 5. Il sequenziamento del Hsp104 Varianti di Colony PCR <ol><li> Raschiare ~ 10 microlitri lievito dalle piastre SD-His-Ura in NaOH 3 ml 20 mM in tubi strip PCR e lisare le cellule di gelo-disgelo. Porre i tubi striscia in un congelatore -80 ° C per 10 min o in azoto liquido, poi incubare a 99 ° C in un termociclatore per 10 min. Diluire i ceppi a 100 ml con acqua di grado PCR. </li><li> Preparare le reazioni PCR: 5 ml modello PCR, 0,5 microlitri 100 mM di ciascun primer, 0,5 ml 10mM dNTP, 0,25 ml polimerasi del DNA in tampone PCR, e portare a 25 ml volume totale di acqua di grado PCR. Primer design per amplificare la regione randomizzato più di circa 100 bp su entrambe le estremità. Minimizzare la dimensione della regione amplificata per aumentare la probabilità di successo di amplificazione. Eseguire un programma standard di PCR. </li><li> Analizzare i campioni mediante elettroforesi su gel di agarosio per confermare l&#39;amplificazione di un prodotto di PCR del siz appropriatae. Ripetere PCR se nessun prodotto è presente. NOTA: il fallimento PCR è in genere a causa di troppo lievito utilizzato nella fase 5.1. </li><li> Preparare i campioni per il sequenziamento del DNA. Rimuovere primer PCR sia mediante PCR di depurazione o uso di un prodotto di PCR pulizia reattivo come esonucleasi I e gamberetti fosfatasi alcalina (ExoSAP-IT) per degradare DNA a singolo filamento. Questo reagente degrada enzimaticamente primer non incorporati e dNTP, consentendo un throughput più elevato. </li><li> Sequenziare i prodotti di PCR. Assicurati di analizzare a fondo cromatogrammi sequenziamento di mutazioni. Le mutazioni possono essere osservati come una miscela di due nucleotidi in un determinato sito (figura 4), ​​come lievito spesso porto più plasmidi. </li></ol>

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The authors have nothing to disclose.

Qui vi presentiamo il nostro approccio per isolare le varianti Hsp104 potenziati che sopprimono la tossicità di substrati associate alla malattia che utilizzano modelli di lievito proteinopathy. Usando questo approccio, grandi librerie di varianti possono essere proiettati in alta velocità, con l&#39;unica limitazione è il numero di varianti che passano schermo secondario 5-FOA. Per eseguire le operazioni descritte in 96 formati bene, abbiamo regolarmente dello schermo fino a 200 colpi alla volta nella fase 5-FOA nel...

Lievito proteinopathy modelli sono stati sviluppati per i disordini di proteine ​​misfolding tra cui la sclerosi laterale amiotrofica (SLA) e la malattia (PD) 1-3 di Parkinson. L&#39;espressione delle proteine ​​TDP-43 e FUS, che misfold nei pazienti con SLA, sono tossici e mislocalize a formare aggregati citoplasmatici in lievito 1,2. Analogamente, l&#39;espressione di α-sinucleina (α-syn), che è implicato nel PD, è tossico e mislocalizes formare aggregati citoplasmatici in lievito 3. Queste caratteristiche ricapitolano fenotipi in pazienti con questi disturbi 4,5. Così, i modelli di lievito forniscono una piattaforma utile per lo screening di proteine ​​o piccole molecole che impediscono o invertire questi fenotipi 2,6-13. Ci interessa lo sviluppo di proteine ​​che sono in grado di invertire aggregazione e tossicità dovuta al TDP-43, FUS, e α-syn. Ci concentriamo su Hsp104, un AAA + proteine ​​da lievito che è l&#39;unica in grado di disaggregare proteine ​​sia fraggregati amorfi OM e amiloide nel lievito, eppure non ha l&#39;omologo umano 14,15. Hsp104 è finemente sintonizzato per disaggregare i prioni di lievito endogene e ha solo limitata capacità di disaggregare substrati implicati nelle malattie neurodegenerative umane, che non ha mai incontra ordinariamente 16,17. Così, ci proponiamo di progettare versioni avanzate di Hsp104 in grado di disaggregare efficacemente questi substrati umani. A tale scopo, costruiamo grandi biblioteche di Hsp104 varianti mediante PCR soggetto a errori; queste librerie possono essere sottoposti a screening utilizzando i modelli di lievito proteinopathy 17. Abbiamo adottato un approccio dominio mirato alla costruzione di screening e biblioteche, come Hsp104 è molto grande 17. Inizialmente concentrati sul dominio centrale (MD) di Hsp104 17, anche se gli approcci simili possono essere impiegati per lo screening altri domini. Questi modelli consentono lo screening per l&#39;attività disaggregase direttamente, al contrario di tecniche alternative come la visualizzazione di superficie, che è riposoricted da utilizzare per il monitoraggio vincolante 18. Il nostro protocollo si basa su due fasi di screening (Figura 1). In primo luogo, vengono selezionati varianti Hsp104 che sopprimono la tossicità del substrato malattia in lievito. Per fare ciò, il Hsp104 varianti e le malattie associate substrato sono cotransformed in lieviti Δ Hsp104. Ci avvaliamo di lievito Hsp104 Δ per esplorare Hsp104 sequenza spazio in assenza di wild-type (WT) Hsp104 17. È importante sottolineare che l&#39;eliminazione di Hsp104 non influisce α-syn, FUS, o TDP-43 della tossicità in lievito, e l&#39;espressione di Hsp104 WT fornisce il minimo soccorso 1,13,17. Il lievito viene poi placcato sulla indurre media per indurre l&#39;espressione di entrambe le proteine. Lievito ospitare varianti Hsp104 che sopprimono la tossicità della crescita substrato conferiscono associata a malattia della colonia. Queste varianti sono state selezionate per ulteriori analisi, mentre le colonie mantenendo le varianti che non sopprimono la tossicità die. Tuttavia,falsi positivi sono un problema sostanziale in questa schermata. Espressione di TDP-43, FUS, e α-syn sono altamente tossici, che crea una forte pressione selettiva per la comparsa di soppressori genetiche spontanee di tossicità correlato alla variante Hsp104 espressi. Così, abbiamo utilizzato uno schermo secondario che è anche relativamente ad alto throughput per eliminare questi soppressori di tossicità non specifici 17. In questa schermata secondaria, lieviti selezionati sono trattati con 5-Fluorootic Acid (5-FOA) per contrastare selezione per il plasmide Hsp104 19. I ceppi sono poi valutati per substrato (TDP-43, FUS o α-syn) tossicità tramite individuazione test per garantire che la tossicità del substrato viene ripristinata dopo la perdita del plasmide Hsp104. Così, lievito in cui tossicità è ripristinato in questa schermata secondaria visualizzata presumibilmente originariamente soppressione tossicità dovuta alla presenza della variante Hsp104. Questi lieviti sono designati come &quot;hit&quot; e il plasmide Hsp104 dovrebbe quindi essererecuperato e sequenziato per identificare le mutazioni nel gene Hsp104 17 (Figura 1). Tutti i colpi devono poi essere riconfermati costruendo la mutazione indipendente utilizzando mutagenesi sito-diretta e poi ripetere il test per la soppressione tossicità. Le potenziali applicazioni di questo protocollo sono ampi. Usando questi metodi, librerie di qualsiasi tipo di proteina potrebbero essere sottoposti a screening per le varianti che sopprimono tossicità di qualsiasi proteina substrato che è tossica in lievito.

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:320px;">Table of Specific Materials/Equipment</td>
			<td style="width:227px;"></td>
			<td style="width:132px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr height="20">
			<td height="20" style="height:20px;">GeneMorphII EZClone Domain Mutagenesis Kit</td>
			<td>Agilent</td>
			<td>200552</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">150mm Petri dishes</td>
			<td>Falcon</td>
			<td>351058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Fluorootic Acid</td>
			<td>Research Products International</td>
			<td>f10501-5.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-DeepWell 2mL Plates</td>
			<td>Eppendorf</td>
			<td>0030 502.302</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 bold replicator tool</td>
			<td>V&#38;P Scientific</td>
			<td>vp-404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ExoSAP-IT</td>
			<td>Affymetrix</td>
			<td>78200</td>
			<td></td>
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</table>

isolating potentiated hsp104 variants using yeast proteinopathy models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Abbiamo costruito una libreria di varianti Hsp104 randomizzati nel dominio centrale e proiettato per la soppressione di TDP-43 tossicità. La biblioteca è stata trasformata e placcato su glucosio e galattosio piastre (Figura 2) per valutare la severità dello schermo. Singole colonie sono stati selezionati e ceppi sono stati selezionati contatore utilizzando 5-FOA per eliminare il plasmide Hsp104. Questi ceppi sono stati poi valutati per confermare che la tossicità era dovuta al TDP-43 da solo, senza ...

Watch this Scientific Journal Video about Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models at JoVE.com

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

L&#39;isolamento potenziato Hsp104 Varianti Utilizzando lievito Proteinopathy modelle

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

isolating-potentiated-hsp104-variants-using-yeast-proteinopathy-models

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.1K Views.  Perelman School of Medicine at the University of Pennsylvania. Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

Video: Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Una valutazione quantitativa del Lipidome lievito con ionizzazione Electrospray Spettrometria di massa

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Ricerca

Biologia

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Studi genetici di proteine ​​umane di riparazione del DNA Utilizzando lievito come sistema modello

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Lievito Colony metodo di inserimento

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

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

Principi di Chirurgia roditori per il chirurgo nuovo

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Contenzione manuale e comuni Itinerari Compound amministrazione in topi e ratti

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

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.

High-throughput iperespressione schermo lievito plasmidi

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

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Purificazione di Hsp104, una proteina Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Crioconservazione del mouse Sperm and Recovery utilizzando il Kit · I Cryo

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

Rilevamento di Rare varianti genomiche da Sequencing pool Uso SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Screening per Funzionale non codificante Varianti genetiche Utilizzando Electrophoretic Mobility Shift Assay (EMSA) e DNA-affinità precipitazioni Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...

L'isolamento potenziato Hsp104 Varianti Utilizzando lievito Proteinopathy modelle

Riepilogo

Esplora Altri Video

Capitoli in questo video

Una valutazione quantitativa del Lipidome lievito con ionizzazione Electrospray Spettrometria di massa

Studi genetici di proteine umane di riparazione del DNA Utilizzando lievito come sistema modello

Lievito Colony metodo di inserimento

Principi di Chirurgia roditori per il chirurgo nuovo

Contenzione manuale e comuni Itinerari Compound amministrazione in topi e ratti

High-throughput iperespressione schermo lievito plasmidi

Purificazione di Hsp104, una proteina Disaggregase

Crioconservazione del mouse Sperm and Recovery utilizzando il Kit · I Cryo

Rilevamento di Rare varianti genomiche da Sequencing pool Uso SPLINTER

Screening per Funzionale non codificante Varianti genetiche Utilizzando Electrophoretic Mobility Shift Assay (EMSA) e DNA-affinità precipitazioni Assay (DAPA)

L'isolamento potenziato Hsp104 Varianti Utilizzando lievito Proteinopathy modelle

Riepilogo

Esplora Altri Video

Capitoli in questo video

Una valutazione quantitativa del Lipidome lievito con ionizzazione Electrospray Spettrometria di massa

Studi genetici di proteine ​​umane di riparazione del DNA Utilizzando lievito come sistema modello

Lievito Colony metodo di inserimento

Principi di Chirurgia roditori per il chirurgo nuovo

Contenzione manuale e comuni Itinerari Compound amministrazione in topi e ratti

High-throughput iperespressione schermo lievito plasmidi

Purificazione di Hsp104, una proteina Disaggregase

Crioconservazione del mouse Sperm and Recovery utilizzando il Kit · I Cryo

Rilevamento di Rare varianti genomiche da Sequencing pool Uso SPLINTER

Screening per Funzionale non codificante Varianti genetiche Utilizzando Electrophoretic Mobility Shift Assay (EMSA) e DNA-affinità precipitazioni Assay (DAPA)

Studi genetici di proteine umane di riparazione del DNA Utilizzando lievito come sistema modello