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. Bibliothèque génération <ol><li> Pour construire des banques de Hsp104 utilisant la PCR sujette à l&#39;erreur spécifique au domaine, d&#39;abord amplifier le domaine d&#39;intérêt avec une erreur de l&#39;ADN polymérase 20 sujets. </li><li> On purifie le produit de PCR par extraction sur gel. </li><li> Effectuez une étape d&#39;extension de méga-utilisant un protocole de mutagenèse dirigée norme: combiner 50 ng modèle plasmide, 250 ng méga-200 uM de dNTP, et haute-fidélité ADN polymérase dans un tampon PCR, et diluer à 50 pi volume total avec PCR eau de qualité 20 . Exécuter un programme PCR standard. REMARQUE: Des amorces spécifiques utilisées varieront en fonction de la région particulière du gène qui doit être amplifié. <ol><li> Suite à la PCR, digérer l&#39;ADN matrice parentale avec 1 ul Dpn I enzyme de restriction pendant 2 heures à 37 ° C. </li></ol></li><li> Transformer la bibliothèque par électroporation ou d&#39;autres moyens de Ultracompetent E. coli et purifier par miniprep. REMARQUE: génération Bibliothèquevarie considérablement sur la base des objectifs d&#39;une expérience donnée. Hsp104 est une très grosse protéine, de sorte qu&#39;il est impossible de rendre aléatoire la totalité du gène, qui est la raison pour laquelle nous utilisons erreur spécifique au domaine PCR sujette. En outre, la structure de Hsp104 reste mal comprise, ce qui rend la conception de bibliothèques destinées difficiles 21. Les bibliothèques peuvent être construites en utilisant des approches dirigées ou aléatoires dans une mutagenèse, avec la seule restriction étant que le modèle de squelette plasmidique doit contenir le gène URA3 pour permettre la 5-FOA sélection de compteur sur un support dextrose. </li></ol> 2. Transformation de la Bibliothèque Hsp104 <ol><li> Intégrer le substrat en W303aΔ Hsp104 levure associée à la maladie en utilisant un acétate de lithium norme / PEG protocole de transformation 22. Sélectionnez des colonies isolées et leur écran de toxicité pour isoler une souche avec une toxicité élevée de la maladie substrat 23 associés. REMARQUE: Utilisez une souche et isola intégréTe une seule colonie d&#39;assurer l&#39;égalité d&#39;expression dans toutes les cellules. Cloner le substrat de la maladie dans un plasmide permettant l&#39;intégration du gène marqueur en vertu d&#39;une autre que l&#39;uracile (nous utilisons histidine) et la bibliothèque Hsp104 dans le plasmide pAG416GAL (marqueur de l&#39;uracile) 24. Pour permettre 5-FOA contre sélection, assurez-vous que la bibliothèque est exprimé à partir d&#39;un plasmide avec le marqueur de l&#39;uracile. D&#39;autres souches de levure peuvent également être employés. Nous avons noté un phénotype de suppression de toxicité similaire en utilisant W303a et BY4741 souches de levures dans les deux milieux de Hsp104 WT et Δ (MEJ et JS, observations non publiées). </li><li> Transformer la bibliothèque Hsp104 dans cette souche en utilisant la même acétate de lithium / PEG protocole de transformation 22. À grande échelle la transformation de façon appropriée pour maintenir l&#39;espace de séquence de la bibliothèque et à préserver la taille de la bibliothèque prédite. </li><li> Plaque du mélange de transformation sur noninducing, plaques sélectives (par exemple, SD-His-Ura) en utilisant assez d&#39;assiettes àassurer la croissance d&#39;un grand nombre de colonies. Utilisation de grandes boîtes de Petri (150 mm) afin de minimiser le nombre de plaques nécessaires. Récupération transformants en utilisant des plaques permet l&#39;efficacité de transformation pour être évalué. </li><li> Transformez Hsp104 WT et vecteur contrôles négatifs en parallèle. </li></ol> 3. Dépistage de la répression des protéotoxicité <ol><li> Laver les colonies au large des plaques en utilisant raffinose complété médias d&#39;abandon (par exemple sraff-His-Ura). Utiliser une pipette sérologique et applicateurs stériles en bois pour desserrer les colonies des plaques. Transférer les liquides de lavage dans un tube conique de 50 ml à fond et vortex pour séparer des amas de cellules. Diluer à une culture légèrement nuageux, DO 600 ~ 0,025. NOTE: Voici sert raffinose pour soulager les cellules de la répression du glucose médiée par l&#39;induction, l&#39;amorçage ainsi les cellules pour l&#39;induction de galactose médiation. </li><li> Cultiver la culture dilué nuit dans un milieu d&#39;abandon raffinose sous agitation à 30 ºC. Cultivez la Hsp104 WT et contrôles de vecteurs en parallèle. </li><li> Le lendemain matin, la plaque une gamme de concentrations SUR DES galactose individu (par exemple SGal-His-Ura) plaques (1 pi à 2 ml concentrés culture dans 400 pi de volume total). Placage une large gamme de concentrations veillera à ce que la plaque aura des colonies isolées. La concentration effective requise dépend de la toxicité du substrat d&#39;intérêt de la maladie. </li><li> Normaliser le vecteur et cultures Hsp104 poids à la DO 600 de la bibliothèque et de la plaque volumes égaux de comparer la croissance de la bibliothèque par rapport aux témoins. </li><li> Pour évaluer sélection rigueur, l&#39;assiette de la bibliothèque sur le glucose (la répression) médias. Incuber les plaques pendant 2-3 jours à 30 ° C, jusqu&#39;à ce que les colonies apparaissent (Figure 2). Évaluer les colonies résultantes et comparer les contrôles. REMARQUE: Souvent, les petites et grandes colonies sont obtenus, mais aucune corrélation entre la taille de la colonie et acteivité est observée. Ecran ces tubes potentiels en utilisant une technique de sélection de compteur 5-FOA (étape 4) afin de minimiser les faux positifs reportés au séquençage. </li></ol> 4. 5-FOA contre sélection et Spotting à éliminer les faux positifs <ol><li> Streak des colonies isolées en double sur support double et simple abandon (par exemple, SD-His-Ura et SD-Ses plaques) et développer une nuit à 30 ° C. Répéter l&#39;opération avec les vecteurs et Hsp104 WT contrôles. Placage sur SD-His avant 5-FOA augmente l&#39;efficacité de l&#39;étape 5-FOA en augmentant la probabilité que les cellules auront perdu le plasmide Hsp104 quand elles sont étalées sur 5-FOA. </li><li> Pour éviter les plaques de sécher, envelopper le SD-ses-Ura plaques de parafilm et conserver à 4 ° C tout en effectuant l&#39;écran 5-FOA. Ces plaques seront éventuellement utilisés pour le séquençage. </li><li> Streak les colonies de la SD-Ses plaques à des colonies isolées sur des plaques 5-FOA (YPD médias + 1g / L 5-FOA) et incuber pendant 1-2 jours à 30 ans &amp;# 186; C jusqu&#39;à ce que des colonies isolées apparaissent. Ici, le 5-FOA est converti en un produit toxique (5-fluorodésoxyuridine) dans les cellules qui contiennent le gène URA3. Ainsi, toutes les cellules encore abritant le plasmide Hsp104 mourront. </li><li> Streak les colonies simples de plaques 5-FOA en double sur une carte SD et SD-Ura-His plaques. Test 3 colonies pour chaque touche pour augmenter la probabilité que au moins une colonie pour chaque coup aura perdu le plasmide Hsp104. Incuber pendant 1-2 jours à 30 ° C. Les colonies qui ont perdu le plasmide Hsp104 va croître sur SD-Ses plaques mais pas les plaques SD-Ura. </li><li> Cultiver les souches qui ont perdu les plasmides Hsp104 pour repérer les essais. Cultiver les souches de la saturation en raffinose médias d&#39;abandon (par exemple sraff-Sa) dans 96 ml DeepWell 2 plaques pendant une nuit à 30 ° C avec agitation. Assurez-vous d&#39;inclure les souches de contrôle 5-FOA traités. </li><li> Préparer les boîtes pour repérer essai. En utilisant une pipette multicanaux, aliquote de 200 pi raffinose médias d&#39;abandon (par exemple sraff-Sa) paret d&#39;une plaque de 96 puits, en réservant les colonnes 1 et 7 pour les cultures soignées. Aliquoter 250 ul de chacune des 16 cultures saturées dans les colonnes 1 et 7 de la plaque. </li><li> Diluer en série les cultures cinq fois dans chaque colonne de la plaque, en mélangeant soigneusement avec une pipette multicanaux. Retirer 50 pi de la culture diluée de colonnes 6 et 12 pour assurer le volume final de chaque puits est de 200 ul. Cela est essentiel pour assurer une spotting. </li><li> L&#39;utilisation d&#39;un outil de réplicateur 96 boulon (Frogger), repérer les cultures en double sur SD-His et SGal-Ses plaques. Incuber les plaques à 30 ° C pendant 2-3 jours. </li><li> Choisir pour le séquençage des souches qui présentent une toxicité sur les SGal-His plaques semblables à celle des témoins. Jeter les souches comme faux positifs qui ne sont pas aussi toxiques que les contrôles. 5-FOA est toxique aussi souvent sur lui-même, de sorte jeter les souches qui présentent un défaut de croissance sur plaques de SD-His ou qui présentent une toxicité nettement supérieure à celle du substrat de la maladie (seuls <strong&gt; Figure 3). </li></ol> 5. Le séquençage du Hsp104 variantes par PCR sur colonie <ol><li> Racler ~ 10 ul de levure à partir des plaques de SD-His-Ura à 3 ul de NaOH 20 mM dans des tubes de PCR en forme de bande et lyser les cellules par congélation-décongélation. Placez les bandes de tubes dans un congélateur -80 ° C pendant 10 minutes ou dans l&#39;azote liquide, puis incuber à 99 ° C dans un thermocycleur pendant 10 min. Diluer les souches à 100 pi avec de l&#39;eau de qualité PCR. </li><li> Préparer les réactions de PCR: 5 pi modèle PCR, 0,5 pi 100 uM de chaque amorce, 0,5 pi 10 mM dNTP, 0,25 ul ADN polymérase dans un tampon PCR, et faire jusqu&#39;à 25 pi volume total de l&#39;eau de qualité PCR. Conception des amorces pour amplifier la région randomisée plus environ 100 pb à chaque extrémité. Réduire la taille de la région amplifiée pour augmenter la probabilité de réussite de l&#39;amplification. Exécuter un programme PCR standard. </li><li> Analyser les échantillons par électrophorèse sur gel d&#39;agarose pour confirmer l&#39;amplification d&#39;un produit de PCR de la siz appropriée. Répétez PCR si aucun produit est présent. REMARQUE: une panne PCR est généralement dû à trop de levure utilisée dans l&#39;étape 5.1. </li><li> Préparation des échantillons pour le séquençage de l&#39;ADN. Retirer des amorces de PCR, soit par purification par PCR ou en utilisant un réactif de nettoyage du produit de PCR telle que l&#39;exonucléase I et l&#39;enzyme phosphatase alcaline de crevette (ExoSAP-IT) pour dégrader l&#39;ADN simple brin. Ce réactif enzymatique dégrade les amorces et les dNTP non constituées en société, permettant un débit plus élevé. </li><li> La séquence des produits de PCR. Assurez-vous de bien analyser chromatogrammes de séquençage des mutations. Des mutations peuvent être observés comme un mélange de deux nucleotides à un site donné (Figure 4), en tant que levure abriter souvent plusieurs plasmides. </li></ol>

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	<li>Johnson, B. S., McCaffery, J. M., Lindquist, S., Gitler, A. 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+yeast+TDP-43+proteinopathy+model:+Exploring+the+molecular+determinants+of+TDP-43+aggregation+and+cellular+toxicity.">A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity.</a> Proc Natl Acad Sci USA. 105 (17), 6439-6444 (2008).</li><li>Sun, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Determinants+and+Genetic+Modifiers+of+Aggregation+and+Toxicity+for+the+ALS+Disease+Protein+FUS/TLS.">Molecular Determinants and Genetic Modifiers of Aggregation and Toxicity for the ALS Disease Protein FUS/TLS.</a> PLoS Biol. 9 (4), (2011).</li><li>Outeiro, T. F., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+Cells+Provide+Insight+into+Alpha-Synuclein+Biology+and+Pathobiology.">Yeast Cells Provide Insight into Alpha-Synuclein Biology and Pathobiology.</a> Science. 302 (5651), 1772-1775 (2003).</li><li>Forman, M. S., Trojanowski, J. Q., Lee, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+diseases:+a+decade+of+discoveries+paves+the+way+for+therapeutic+breakthroughs.">Neurodegenerative diseases: a decade of discoveries paves the way for therapeutic breakthroughs.</a> Nat Med. 10 (10), 1055-1063 (2004).</li><li>Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress,+aging,+and+neurodegenerative+disease.">Stress, aging, and neurodegenerative disease.</a> N Engl J Med. 355 (21), 2254-2255 (2006).</li><li>Elden, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ataxin-2+intermediate-length+polyglutamine+expansions+are+associated+with+increased+risk+for+ALS.">Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.</a> Nature. 466 (7310), 1069-1075 (2010).</li><li>Cooper, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+blocks+ER-Golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson's+models.">Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313 (5785), 324-328 (2006).</li><li>Gitler, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-synuclein+is+part+of+a+diverse+and+highly+conserved+interaction+network+that+includes+PARK9+and+manganese+toxicity.">Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity.</a> Nature genetics. 41 (3), 308-315 (2009).</li><li>Su, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compounds+from+an+unbiased+chemical+screen+reverse+both+ER-to-Golgi+trafficking+defects+and+mitochondrial+dysfunction+in+Parkinson's+disease+models.">Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.</a> Disease models &amp; mechanisms. (3-4), 194-208 (2010).</li><li>Tardiff, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+reveal+a+'druggable'+Rsp5/Nedd4+network+that+ameliorates+alpha-synuclein+toxicity+in+neurons.">Yeast reveal a 'druggable' Rsp5/Nedd4 network that ameliorates alpha-synuclein toxicity in neurons.</a> Science. 342 (6161), 979-983 (2013).</li><li>Tardiff, D. F., Tucci, M. L., Caldwell, K. A., Caldwell, G. A., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+8-hydroxyquinolines+protect+models+of+TDP-43+protein,+alpha-synuclein,+and+polyglutamine+proteotoxicity+through+distinct+mechanisms.">Different 8-hydroxyquinolines protect models of TDP-43 protein, alpha-synuclein, and polyglutamine proteotoxicity through distinct mechanisms.</a> The Journal of biological chemistry. 287 (6), 4107-4120 (2012).</li><li>Kim, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+modulation+of+eIF2alpha+phosphorylation+rescues+TDP-43+toxicity+in+amyotrophic+lateral+sclerosis+disease+models.">Therapeutic modulation of eIF2alpha phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models.</a> Nature. 46 (2), 152-160 (2014).</li><li>Ju, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+model+of+FUS/TLS-dependent+cytotoxicity.">A yeast model of FUS/TLS-dependent cytotoxicity.</a> PLoS Biol. 9 (4), (2011).</li><li>Shorter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp104:+a+weapon+to+combat+diverse+neurodegenerative+disorders.">Hsp104: a weapon to combat diverse neurodegenerative disorders.</a> Neurosignals. 16 (1), 63-74 (2008).</li><li>Vashist, S., Cushman, M., Shorter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+Hsp104+to+protein-misfolding+disorders.">Applying Hsp104 to protein-misfolding disorders.</a> Biochem Cell Biol. 88 (1), 1-13 (2010).</li><li>DeSantis, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operational+Plasticity+Enables+Hsp104+to+Disaggregate+Diverse+Amyloid+and+Nonamyloid+Clients.">Operational Plasticity Enables Hsp104 to Disaggregate Diverse Amyloid and Nonamyloid Clients.</a> Cell. 151 (4), 778-793 (2012).</li><li>Jackrel, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiated+Hsp104+Variants+Antagonize+Diverse+Proteotoxic+Misfolding+Events.">Potentiated Hsp104 Variants Antagonize Diverse Proteotoxic Misfolding Events.</a> Cell. 156 (1-2), 170-182 (2014).</li><li>Wittrup, K. D., Verdine, G. L., Wittrup, K. D., Verdine, G. L. <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 Enzymology. 503, 13-14 (2012).</li><li>Boeke, J. D., Trueheart, J., Natsoulis, G., Fink, G. R. <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 Enzymology. 154, 164-175 (1987).</li><li>Miyazaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+Random+Mutagenesis+Libraries+by+Megaprimer+PCR+of+Whole+Plasmid+(MEGAWHOP).+Directed+Evolution+Library+Creation:+Methods+in+Molecular+Biology.">Creating Random Mutagenesis Libraries by Megaprimer PCR of Whole Plasmid (MEGAWHOP). Directed Evolution Library Creation: Methods in Molecular Biology.</a> eds FrancesH Arnold &amp; George Georgiou. 231, 23-28 (2003).</li><li>Saibil, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperone+machines+for+protein+folding,+unfolding+and+disaggregation.">Chaperone machines for protein folding, unfolding and disaggregation.</a> Nat Rev Mol Cell Biol. 14 (10), 630-642 (2013).</li><li>Gietz, R. D., Schiestl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+yeast+transformation+using+the+LiAc/SS+carrier+DNA/PEG+method.">High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method.</a> Nat. Protocols. 2 (1), 31-34 (2007).</li><li>Armakola, M., Hart, M. P., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TDP-43+toxicity+in+yeast.">TDP-43 toxicity in yeast.</a> Methods. 53 (3), 238-245 (2011).</li><li>Alberti, S., Gitler, A. D., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+suite+of+Gateway&#174;+cloning+vectors+for+high-throughput+genetic+analysis+in+Saccharomyces+cerevisiae.">A suite of Gateway&#174; cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae.</a> Yeast. 24 (10), 913-919 (2007).</li></ol>

The authors have nothing to disclose.

Ici, nous présentons notre approche pour isoler variantes Hsp104 potentialisés qui suppriment la toxicité des substrats associés à la maladie en utilisant des modèles de levure proteinopathy. En utilisant cette approche, les grandes banques de variants peuvent être criblés en haut débit, avec la seule limitation étant le nombre de variantes qui passent l&#39;écran secondaire 5-FOA. En effectuant ces mesures au format 96 puits, nous contrôlons régulièrement jusqu&#39;à 200 coups à la fois dans l&#39;étap...

Levure de modèles proteinopathy ont été développés pour les troubles du repliement des protéines, y compris la sclérose latérale amyotrophique (SLA) et la maladie (PD) 3/1 de Parkinson. L&#39;expression des protéines TDP-43 et FUS, qui misfold chez les patients SLA, sont toxiques et mislocalize à former des agrégats cytoplasmiques dans la levure 1,2. De même, l&#39;expression d&#39;α-synucléine (α-syn), qui est impliquée dans la MP, est toxique et mislocalizes à former des agrégats cytoplasmiques de levure 3. Ces caractéristiques récapitulent phénotypes chez les patients atteints de ces troubles 4,5. Ainsi, les modèles de levure fournissent une plate-forme utile pour le criblage de protéines ou de petites molécules qui empêchent ou inverser ces phénotypes 2,6-13. Nous sommes intéressés par le développement de protéines qui sont capables de renverser l&#39;agrégation et la toxicité due à TDP-43, FUS, et α-syn. Nous nous concentrons sur Hsp104, un AAA + protéines de la levure qui est uniquement capable de désagréger la fois des protéines fragrégats amorphes l&#39;OM et l&#39;amyloïde dans la levure, mais il n&#39;a pas homologue humain 14,15. Hsp104 est finement réglé pour désagréger les prions de levure endogènes et ne dispose que d&#39;une capacité limitée à ventiler substrats impliqués dans les maladies neurodégénératives humaines, qu&#39;il rencontre jamais normalement 16,17. Ainsi, nous avons pour objectif de concevoir des versions améliorées de Hsp104 qui sont en mesure de ventiler efficacement ces substrats humains. Pour ce faire, nous construisons les grandes bibliothèques de variantes Hsp104 par PCR sujette à l&#39;erreur; Ces bibliothèques peuvent être contrôlés au moyen des modèles de levure proteinopathy 17. Nous avons adopté une approche de domaine ciblé pour la construction et le criblage de banques, comme Hsp104 est très grand 17. Nous avons d&#39;abord mis l&#39;accent sur ​​le domaine du milieu (MD) de Hsp104 17, même si les approches similaires peuvent être utilisés pour dépister d&#39;autres domaines. Ces modèles permettent de cribler l&#39;activité de disaggregase directement, par opposition à d&#39;autres techniques telles que l&#39;affichage de la surface, qui est le reposricted à utiliser pour la surveillance de la liaison 18. Notre protocole est basé sur deux étapes de criblage (Figure 1). Tout d&#39;abord, Hsp104 variantes qui suppriment la toxicité du substrat de la maladie chez la levure sont sélectionnées. Pour ce faire, le Hsp104 variantes et associée à la maladie substrat sont cotransformé dans Δ Hsp104 levure. Nous employons Δ Hsp104 levure à explorer Hsp104 espace de séquence en l&#39;absence de type sauvage (WT) Hsp104 17. Fait important, la deletion de Hsp104 n&#39;a pas d&#39;incidence α-syn, FUS, ou TDP-43 toxicité dans la levure, et l&#39;expression de Hsp104 WT prévoit un minimum de sauvetage 1,13,17. La levure est ensuite étalé sur des milieux induisant pour induire l&#39;expression des deux protéines. Levure hébergeant Hsp104 variantes qui suppriment la toxicité de la croissance de la colonie confèrent substrat associé à une maladie. Ces variantes sont sélectionnées pour une analyse plus approfondie, tandis que le maintien de colonies variantes qui ne suppriment pas la toxicité dé. Cependant,les faux positifs sont un problème important dans cet écran. L&#39;expression de TDP-43, FUS, et α-syn sont hautement toxiques, ce qui crée une forte pression de sélection pour l&#39;apparition de suppresseurs génétiques spontanées de toxicité sans rapport avec la variante Hsp104 être exprimé. Ainsi, nous avons utilisé un écran secondaire qui est aussi relativement haut débit pour éliminer ces suppresseurs de toxicité non spécifiques 17. Dans cet écran secondaire, levures sélectionnées sont traités avec 5 Fluorootic acide (5-FOA) pour contrer sélection pour le plasmide Hsp104 19. Les souches sont ensuite évaluées pour substrat (TDP-43, FUS, ou α-syn) toxicité par analyse des taches de sorte que la toxicité du substrat est rétablie après la perte du plasmide Hsp104. Ainsi, la levure, dans lequel la toxicité est restaurée dans l&#39;écran secondaire affiche initialement doute suppression de la toxicité due à la présence du variant Hsp104. Ces levures sont désignés comme «hits» et le plasmide Hsp104 devrait alors êtrerécupérée et séquences pour identifier les mutations dans le gène Hsp104 17 (figure 1). Les résultats doivent ensuite être confirmées par la construction de la mutation indépendamment en utilisant la mutagenèse dirigée et retester pour la suppression de toxicité alors. Les applications potentielles de ce protocole sont larges. En utilisant ces procédés, des banques de tout type de protéine peuvent être criblés pour des variantes qui suppriment la toxicité d&#39;une protéine substrat qui est toxique à la levure.

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			<td height="20" style="height:20px;width:320px;">Table of Specific Materials/Equipment</td>
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			<td style="width:87px;"></td>
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			<td height="20" style="height:20px;">Name</td>
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			<td>Catalog Number</td>
			<td>Comments</td>
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			<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>
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			<td height="20" style="height:20px;">5-Fluorootic Acid</td>
			<td>Research Products International</td>
			<td>f10501-5.0</td>
			<td></td>
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			<td height="20" style="height:20px;">96-DeepWell 2mL Plates</td>
			<td>Eppendorf</td>
			<td>0030 502.302</td>
			<td></td>
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		<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>
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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.

Nous avons construit une banque de variants Hsp104 randomisés dans le domaine intermédiaire et criblés pour la suppression de TDP-43 toxicité. La bibliothèque a été transformée et plaqué sur du glucose et du galactose plaques (figure 2) pour évaluer la rigueur de l&#39;écran. Des colonies isolées ont été sélectionnés et les souches ont été sélectionnées à l&#39;aide contre 5-FOA pour éliminer le plasmide Hsp104. Ces souches ont ensuite été évalués pour confirmer que la toxicit?...

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.

Isoler Potentiated Hsp104 Variantes utilisant de la levure Proteinopathy modèles

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

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Biology

8.1K vues.  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.

Vidéo: 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.

Une évaluation quantitative du lipidome levure en utilisant la spectrométrie de masse électrospray ionisation

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

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Biologie

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.

Les études génétiques des protéines humaines réparation de l&#39;ADN utilisant de la levure comme système modèle

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.

Méthode d&#39;insertion de levure 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.

Principes de la chirurgie des rongeurs pour le nouveau chirurgien

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.

Manuel de retenue et communes voies d&#39;administration composés chez la souris et le rat

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.

Haut débit écran surexpression plasmide de levure

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.

Purification de Hsp104, une protéine 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.

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.

Cryoconservation du sperme de souris et de récupération utilisant les 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.

Détection de rares variantes génomiques à partir de séquençage regroupés à l&#39;aide 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.

Le dépistage de la fonctionnelle non-codant des variants génétiques utilisant mobilité électrophorétique Maj Assay (EMSA) et l&#39;ADN-affinité Précipitations Assay (DAPA)

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