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> Para construir bibliotecas de Hsp104 usando PCR propensa a erros de domínio específico, primeiro amplificar o domínio de interesse com um erro DNA polimerase propensas 20. </li><li> Purifica-se o produto de PCR por extracção de gel. </li><li> Realizar uma etapa de extensão megainiciador utilizando um protocolo padrão de mutagénese dirigida ao local: combinar 50 ng de plasmídeo, modelo, 250 megainiciador ng, 200 uM de dNTPs, e de alta fidelidade da polimerase de ADN em tampão de PCR, e diluir até 50 mL de volume total, com água de grau de PCR 20 . Execute um programa de PCR padrão. NOTA: Os primers específicos utilizados irá variar com base na região particular do gene que está a ser amplificado. <ol><li> Após a PCR, digerir ADN molde parental com 1 ul da enzima de restrição Dpnl durante 2 horas a 37 ° C. </li></ol></li><li> Transformar a biblioteca por eletroporação ou outros meios em ultracompetent E. coli e purificá-lo por miniprep. NOTA: geração Bibliotecavaria substancialmente com base nos objetivos de um determinado experimento. Hsp104 é uma proteína muito grande, por isso é impraticável para embaralhar todo o gene, que é por isso que nós utilizamos erro específico do domínio propenso PCR. Além disso, a estrutura de Hsp104 permanece pouco compreendido, tornando o desenho de bibliotecas dirigidas 21 desafiantes. As bibliotecas podem ser construídas utilizando abordagens dirigidas ou aleatórias para mutagénese, com a única restrição que o plasmídeo de esqueleto modelo deve conter o gene URA3 para permitir 5-FOA contra-selecção em meios de dextrose. </li></ol> 2. Transformação da Biblioteca Hsp104 <ol><li> Integrar o substrato associado à doença em W303aΔ Hsp104 levedura usando um acetato de lítio padrão / PEG protocolo de transformação 22. Selecione colónias individuais e analisá-los em termos de toxicidade para isolar uma cepa com alta toxicidade da doença associada substrato 23. NOTA: Use uma tensão integrado e isolate uma única colônia de garantir a igualdade de expressão em todas as células. Clonar o substrato de doença num plasmídeo que permite a integração do gene em qualquer outro marcador de uracilo (usamos histidina) e a biblioteca de Hsp104 no plasmídeo pAG416GAL (marcador uracilo) 24. Para permitir o 5-FOA counterselection, assegurar que a biblioteca é expressa a partir de um plasmídeo com a marca de uracilo. Outras estirpes de levedura também podem ser empregues. Temos notado um fenótipo supressão toxicidade semelhante usando W303a e BY4741 cepas de leveduras em ambos WT e Δ Hsp104 fundos (MEJ e JS, observações não publicadas). </li><li> Transformar a biblioteca em Hsp104 esta estirpe usando o mesmo acetato de lítio / PEG protocolo de transformação 22. Dimensionar-se a transformação de forma adequada para manter o espaço de sequência da biblioteca e para preservar o tamanho da biblioteca previsto. </li><li> Placa da mistura de transformação para noninducing, placas seletivas (por exemplo SD-His-Ura) usando placas suficientes paraassegurar o crescimento de um grande número de colónias. Use grandes placas de Petri (150 mm) para minimizar o número de placas necessárias. Recuperando transformantes utilizando placas permite eficiência de transformação para ser avaliado. </li><li> Transforme Hsp104 WT e controles negativos vetor em paralelo. </li></ol> 3. Triagem para Supressão de Proteotoxicity <ol><li> Lave as colônias fora as placas usando rafinose complementada media de abandono (por exemplo SRaff-His-URA). Usar uma pipeta serológica estéril e aplicadores de madeira para soltar as colónias das placas. Transferir as lavagens líquidos para um tubo cónico de 50 ml e cuidadosamente vórtice para separar quaisquer aglomerações de células. Diluir a uma cultura um pouco nublado, OD 600 ~ 0,025. NOTA: Aqui rafinose serve para aliviar as células de repressão mediada por glicose de indução, priming assim que as células de indução mediada por galactose. </li><li> Incubar a cultura diluído durante a noite em meio de abandono rafinose com agitação a 30 ºC. Crescer a Hsp104 WT e controle de vetores em paralelo. </li><li> Na manhã seguinte, chapear uma gama de concentrações de galactose Onto individual (por exemplo SGAL-His-Ura) placas (1 ul a 2 ml da cultura concentradas em 400 ul de volume total). Chapeamento de uma ampla gama de concentrações vai garantir que uma placa vai ter colónias isoladas. A concentração real necessário depende da toxicidade do substrato doença de interesse. </li><li> Normalizar o vector e culturas Hsp104 WT para o OD 600 da biblioteca e placa volumes iguais para comparar o crescimento da biblioteca em relação aos controlos. </li><li> Para avaliar a seleção rigor, placa da biblioteca em glicose (reprimir) mídia. Incubar as placas durante 2-3 dias a 30 ° C, até que colónias aparecem (Figura 2). Avaliar as colônias resultantes e comparar com os controles. NOTA: Muitas vezes pequenos e grandes colônias são obtidos, mas nenhuma correlação entre o tamanho da colônia e agirivity é observado. Tela estes potenciais sucessos usando uma técnica de contra-selecção 5-FOA (Passo 4) para minimizar os falsos positivos transitados para sequenciação. </li></ol> 4. 5-FOA Counterselection e Spotting para eliminar falsos positivos <ol><li> Streak fora colónias isoladas em duplicata em suporte de casal e solteiro abandono (por exemplo SD-His-Ura e-Seus SD placas) e crescer durante a noite a 30 ºC. Repita a operação com controle de vetores e Hsp104 WT. Plaqueamento em SD-His antes da 5-FOA aumenta a eficiência do passo 5-FOA, aumentando a probabilidade de que as células terão perdido o plasmídeo Hsp104 quando são semeadas em 5-FOA. </li><li> Para evitar que as placas de secar, enrole a placas em parafilm Seus-SD-Ura e armazenar a 4 ºC durante a realização da tela de 5 FOA. Estas placas venham a ser utilizados para a sequenciação. </li><li> Streak as colônias do SD-Suas placas de colónias isoladas em placas 5-FOA (YPD media + 1g / L 5-FOA) e incubar durante 1-2 dias a 30 &amp;# 186; C até as colónias individuais aparecem. Aqui, 5-FOA é convertido num produto tóxico (5-fluorodesoxiuridina) em células que contêm o gene URA3. Assim, destacando as células portadoras do plasmídeo Hsp104 morrerá. </li><li> Streak as colónias individuais das placas 5-FOA em duplicar em SD-Ura e-Seus SD placas. Teste 3 colónias para cada batida para aumentar a probabilidade de que pelo menos uma colónia de cada um batido terá perdido o plasmídeo de Hsp104. Incubar durante 1-2 dias a 30 ° C. As colónias que perderam o plasmídeo Hsp104 vai crescer em SD-His placas mas não placas de SD-Ura. </li><li> Crescer as tensões que perderam os plasmídeos Hsp104 para detectar ensaios. Crescer as tensões à saturação em rafinose mídia abandono (por exemplo SRaff-His) em 96 Deepwell 2 placas ml durante a noite a 30 ºC com agitação. Certifique-se de incluir as estirpes de controlo 5-FOA tratados. </li><li> Prepare placas para detectar ensaio. Usando uma pipeta de canais múltiplos, alíquota de 200 ul rafinose meios de abandono (por exemplo SRaff-His) porpoço de uma placa de 96 poços, reservando colunas 1 e 7 para as culturas puras. Alíquota de 250 mL de cada uma das 16 culturas saturadas em colunas 1 e 7 da placa. </li><li> Serialmente dilua as culturas de 5 vezes em cada coluna da placa, misturar cuidadosamente com uma pipeta de canais múltiplos. Retirar 50 mL da cultura diluída a partir de colunas 6 e 12 para garantir o volume final de cada poço é 200 ul. Isto é essencial para garantir uma identificação. </li><li> Usando uma ferramenta replicador de 96 parafuso (Frogger), identificar as culturas em duplicado para-Seus SD e SGAL-Seus pratos. Incubar as placas a 30 ºC durante 2-3 dias. </li><li> Selecione para sequenciar cepas que apresentam toxicidade nas SGAL-Suas placas semelhantes aos dos controles. Descarte cepas como falsos positivos que não são tão tóxico como os controles. 5-FOA é também muitas vezes tóxico por si próprio, então descartar cepas que apresentam um defeito de crescimento em-seu SD ou placas que exibem substancialmente maior toxicidade do que o substrato doença sozinho ( <strong&gt; A Figura 3). </li></ol> 5. O seqüenciamento do Hsp104 Variantes por Colony PCR <ol><li> Raspe ~ 10 ul de levedura a partir das placas de SD-His-Ura em NaOH 3 ul 20 mM em tubos de PCR de tira e lisar as células por congelação-descongelação. Colocar os tubos em um congelador tira -80 ºC durante 10 min ou em azoto líquido, depois incubar a 99 ° C num termociclador durante 10 min. Dilui-se as estirpes para 100 ul com água de grau de PCR. </li><li> Prepare reações de PCR: 5 jul modelo PCR, 0,5 ul 100? M de cada iniciador, 0,5 mL de 10 mM dNTPs, 0,25 mL DNA polimerase em tampão PCR, e fazer até 25 mL volume total com PCR água grau. Design Os iniciadores para amplificar a região mais ao acaso, aproximadamente 100 pb em cada extremidade. Minimizar o tamanho da região amplificada para aumentar a probabilidade de uma amplificação bem sucedida. Execute um programa de PCR padrão. </li><li> Analisar amostras por electroforese em gel de agarose para confirmar a amplificação de um produto de PCR do siz adequadoe. Repita PCR se nenhum produto está presente. NOTA: A falha PCR é geralmente devido ao excesso de levedura a ser utilizado na etapa 5.1. </li><li> Preparar amostras para seqüenciamento de DNA. Remover os iniciadores de PCR quer através de purificação de PCR ou utilizando um reagente de limpeza do produto de PCR, tais como a Exonuclease I e fosfatase alcalina de camarão enzima (ExoSAP-TI) para degradar o ADN de cadeia simples. Este reagente enzimático degrada primers não incorporados e dNTPs, permitindo maior rendimento. </li><li> Sequenciar os produtos de PCR. Certifique-se de analisar cuidadosamente cromatogramas de sequenciamento de mutações. As mutações podem ser observadas como uma mistura de dois nucleótidos em um dado local (Figura 4), ​​como de levedura muitas vezes abrigam os plasmídeos múltiplos. </li></ol>

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The authors have nothing to disclose.

Aqui apresentamos nossa abordagem para isolar variantes Hsp104 potencializadas que suprimem a toxicidade de substratos associados à doença, utilizando modelos de levedura proteinopathy. Utilizando esta abordagem, bibliotecas grandes de variantes pode ser rastreada em elevado rendimento, com a única limitação é o número de variantes que passam o ecrã secundário 5-FOA. Ao realizar estes passos em formato de 96 poços, que rotineiramente tela até 200 batidas de cada vez no passo 5-FOA ao longo de 1-2 semanas. O n...

Levedura proteinopathy modelos têm sido desenvolvidos para distúrbios-misfolding proteína incluindo a esclerose lateral amiotrófica (ELA) e a doença de Parkinson (DP) 1-3. Expressão das proteínas TDP-43 e FUS, que misfold em pacientes de ELA, são tóxicos e mislocalize para formar agregados citoplasmáticos em levedura 1,2. Do mesmo modo, a expressão de α-sinucleína (α-syn), que está implicado na doença de Parkinson, é tóxico e mislocalizes para formar agregados citoplasmáticos em 3 de levedura. Esses recursos recapitular fenótipos em pacientes com esses transtornos 4,5. Assim, os modelos de levedura fornecer uma plataforma útil para o rastreio de proteínas ou moléculas pequenas que impedem ou invertem estes fenótipos 2,6-13. Estamos interessados ​​no desenvolvimento de proteínas que são capazes de inverter a agregação e toxicidade devido a TDP-43, FUS, e α-syn. Nós nos concentramos em Hsp104, um AAA + proteína de levedura que é o único capaz de desagregar proteínas tanto fragregados amorfos om e amilóide em leveduras, ainda não tem homólogo humano 14,15. Hsp104 é afinado para desagregar prions levedura endógenos e tem apenas capacidade limitada para desagregar substratos implicados em doenças neurodegenerativas humanas, que nunca encontra ordinariamente 16,17. Assim, nosso objetivo é projetar versões aprimoradas de Hsp104 que são capazes de desagregar eficazmente estes substratos humanos. Para isso, construímos grandes bibliotecas de Hsp104 variantes utilizando PCR propenso a erros; essas bibliotecas podem ser rastreados usando os modelos de levedura proteinopathy 17. Adotamos uma abordagem orientada-domínio para construção e pesquisa de bibliotecas, como Hsp104 é muito grande 17. Nós centrou-se inicialmente no domínio médio (DM) de Hsp104 17, embora as abordagens semelhantes podem ser empregues para o rastreio de outros domínios. Esses modelos permitem o rastreio de actividade disaggregase directamente, ao contrário de técnicas alternativas, tais como exposição de superfície, que é restoricted usar para monitorar a ligação 18. O protocolo baseia-se em dois passos de triagem (Figura 1). Em primeiro lugar, as variantes de Hsp104 que suprimem a toxicidade do substrato doença em levedura são seleccionados. Para fazê-lo, a Hsp104 variantes e associado à doença substrato são co-transformados em levedura Δ Hsp104. Nós empregamos Δ Hsp104 levedura para explorar Hsp104 seqüência espaço na ausência de tipo selvagem (WT) Hsp104 17. Importante, supressão de Hsp104 não afecta α-syn, FUS, ou TDP-43 toxicidade em levedura, e expressão de Hsp104 WT fornece salvamento mínima 1,13,17. A levedura é então plaqueadas em meios de indução para induzir a expressão de ambas as proteínas. Levedura abrigar variantes Hsp104 que suprimem a toxicidade do associado à doença de crescimento substrato conferem da colônia. Estas variantes são seleccionados para posterior análise, mantendo ao mesmo tempo as colónias variantes que não suprimem a toxicidade da matriz. No entanto,falsos positivos são um problema substancial na tela. Expressão de TDP-43, FUS, e α-syn são altamente tóxicos, o que cria uma forte pressão selectiva para o aparecimento de supressores genéticas espontâneas de toxicidade relacionada com a variante Hsp104 ser expresso. Assim, temos utilizado uma tela secundária que também é relativamente alto rendimento para eliminar esses supressores de toxicidade inespecíficos 17. Nesta tela secundária, leveduras selecionadas são tratados com 5-Fluorootic ácido (5-FOA) para combater a seleção para o plasmídeo Hsp104 19. As estirpes são então avaliadas para substrato (TDP-43, FUS, ou α-sin) toxicidade via ensaio de mancha para assegurar que a toxicidade do substrato é restaurada após a perda do plasmídeo Hsp104. Assim, de levedura, em que a toxicidade é restaurada neste ecrã secundário originalmente apresentado presumivelmente supressão de toxicidade devido à presença da variante da Hsp104. Estas leveduras são designados como &#39;hits&#39; e do plasmídeo Hsp104 deve, então, serrecuperados e sequenciados para identificar as mutações no gene de Hsp104 17 (Figura 1). Qualquer resultado deve ser reconfirmada em seguida, através da construção da mutação, independentemente, utilizando mutagénese dirigida ao local e, em seguida, para a supressão de novo teste de toxicidade. As potenciais aplicações para este protocolo são amplas. Usando estes métodos, bibliotecas de qualquer tipo de proteína pode ser rastreada para as variantes que suprimem a toxicidade da proteína de qualquer substrato que é tóxico em levedura.

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

Construímos uma biblioteca de variantes de Hsp104 randomizados no domínio médio e rastreados-lo para a supressão de TDP-43 toxicidade. A biblioteca foi transformada e plaqueada em placas de glicose e galactose (Figura 2) para avaliar a severidade da tela. Colónias individuais foram seleccionadas e as estirpes foram contra-seleccionadas utilizando 5-FOA para eliminar o plasmídeo Hsp104. Estas estirpes foram então avaliadas para confirmar que a toxicidade foi devido a TDP-43 sozinho, sem as variant...

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.

Isolando potencializado Hsp104 variantes usando levedura 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 ...

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

Research

JoVE Journal

Biology

8.1K visualizações.  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.

Vídeo: 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.

A avaliação quantitativa do Lipidome fermento usando Electrospray Espectrometria de Massa de Ionização

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

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.

Estudos genéticos de proteínas de reparo do DNA Humano Utilizando Levedura como um sistema modelo

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.

Levedura Método Embedding Colony

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.

Princípios de Cirurgia de roedores para o Cirurgião Nova

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.

Restrição manual e rotas de Administração composto comum em camundongos e ratos

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.

Alto rendimento Yeast Tela superexpressão Plasmid

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.

Purificação da Hsp104, um Disaggregase Protein

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.

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

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.

Criopreservação do mouse Sperm and Recovery utilizando o I · 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 ...

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.

Detecção de variantes raras genômico de Seqüenciamento combinados usando 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.

Triagem para Funcional não-codificante variantes genéticas Usando eletroforética Mobilidade Mudança Assay (EMSA) e DNA afinidade Precipitation Assay (DAPA)

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