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. Kütüphane Oluşturma <ol><li> Domain-spesifik hata eğilimli PCR kullanarak Hsp104 kütüphaneleri oluşturmak için, ilk önce bir hata eğilimli DNA polimeraz 20 ile ilgi alanını yükseltmek. </li><li> Jel ekstre PCR ürünü saflaştınlır. </li><li> Bir standart alana-yönlendirilmiş mutajenez protokolü kullanılarak bir megaprimer uzatma adımı gerçekleştirmek: PCR tamponu içinde 50 ng şablon plazmiti, 250 ng megaprimer, 200 uM dNTP, ve yüksek doğrulukta bir DNA polimerazı birleştirmek, ve PCR dereceli su 20, 50 ul toplam hacme kadar seyreltin . Standart bir PCR programı çalıştırın. Not: amplifiye edilecek olan genin özel bir bölgesine göre değişir, kullanılan spesifik primerler. <ol><li> PCR sonra, 37 ° C&#39;de 2 saat süre ile, 1 ul Dpn I sınırlama enzimi ile ebeveyn hedef DNA sindirimi. </li></ol></li><li> Ultracompetent E. elektroporasyon veya diğer yollarla kitaplığı Transform coli Miniprep bunu arındırmak ve. NOT: Kütüphane oluşturmaesas olarak, belirli bir deney amaçlarına göre değişir. Hsp104 çok büyük bir protein olduğu, bu yüzden biz alanı özel hata eğilimli PCR kullanmak neden bütün geni, rastgele pratik değildir. Buna ek olarak, Hsp104 yapısı az 21 zorlu, yönlendirilmiş kitaplıkların tasarım, yapım anlaşılamamıştır. Kütüphane tek sınırlama, şablon plazmid omurgası dekstroz ortama 5-FOA karşı seçilmesine olanak sağlamak için, URA3 geni ihtiva etmesi olması ile, mutajenez için yönlendirilmiş veya rasgele yaklaşımlar kullanılarak yapılabilir. </li></ol> Hsp104 Kütüphanesi 2. Dönüşüm <ol><li> Standart bir lityum asetat / PEG transformasyon protokolü ile 22 W303aΔ hsp104 mayaya hastalıkla ilişkili alt-tabakanın entegre. Tek koloniler seçmek ve alt-tabakanın 23 ilgili hastalığın yüksek bir toksisite ile suşu izole toksisite için onları ekran. NOT: entegre bir gerginlik ve isola kullanınTüm hücreler eşit ekspresyonunu sağlamak için tek bir koloni te. PAG416GAL plazmid (urasil belirteç) 24 içine urasil dışında herhangi bir marker (biz histidin kullanın) ve Hsp104 kütüphane altında gen entegrasyonunu sağlayan bir plazmide hastalık tabakayı Clone. 5-FOA ters seçim olanak sağlamak için, kitaplık urasil markerinde bir plasmitten ifade olduğundan emin olun. Diğer maya suşları da kullanılabilir. Biz W303a hem WT ve Δ hsp104 kökenden BY4741 maya suşları (MEJ ve JS, yayınlanmamış gözlemler) kullanarak benzer bir toksisite bastırma fenotip kaydetti. </li><li> Aynı lityum asetat / PEG transformasyon protokolü 22 kullanarak bu tür haline Hsp104 kitaplığı dönüşümü. Kütüphanenin dizisi alanı muhafaza etmek için ve tahmin edilen kitaplık ebadını korumak için uygun dönüşümü büyütmek. </li><li> Kadar plakalar kullanılarak noninducing, seçici plakalar üzerine transformasyon karışımı (örneğin, SD-His-Ura) plakakoloniler, çok sayıda büyümesini sağlar. Gerekli plakaların sayısını en aza indirmek için büyük petri kapları (150 mm) kullanın. Plakalar kullanılarak transformantların kurtarma dönüşüm etkinliği değerlendirilir sağlar. </li><li> Paralel Hsp104 WT ve vektör negatif kontrolleri Transform. </li></ol> Proteotoxicity ve Önlenmesine 3. Eleme <ol><li> Rafınoz takviye bırakma ortamı (örn sraff-His-Ura) kullanarak plakaları kapalı koloniler yıkayın. Plakalardan koloniler gevşetmek için bir serolojik pipet ve steril ahşap aplikatörler kullanın. 50 ml konik bir tüp ve vorteks sıvı yıkama transfer iyice hücrelerin herhangi bir yığınının ayrı. Biraz bulutlu kültür, OD 600 ~ 0.025 sulandırmak. Not: Burada rafinoz ve böylece galaktoz aracılı indüksiyon hücreleri priming, indüksiyon glukoz aracılı baskı hücrelerini azaltmak için hizmet eder. </li><li> 3 çalkalanarak rafınoz bırakma medyada bir gecede seyreltilmiş kültür büyütün0 ºC. Paralel Hsp104 WT ve vektör kontrol büyür. </li><li> Sonraki sabah, tek tek galaktoz üzerine konsantrasyonları bir dizi plaka (örneğin SGAL-His-Ura) plakaları (400 ul toplam hacim içinde kültür konsantre edildi 2 ml için 1 ul). Konsantrasyonlarının geniş bir kaplama bir plaka tek koloniler sahip sağlayacaktır. Gerekli gerçek konsantrasyon yararlı hastalık substratın toksisite bağlıdır. </li><li> Kütüphanenin OD 600 vektör ve Hsp104 WT kültürleri normalize ve kontrol kütüphane nisbetle büyümesini karşılaştırma eşit hacimlerde plaka. </li><li> Seçim sertliğini değerlendirmek için, medya (bastırma) glukoz kütüphane plaka. Koloniler (Şekil 2) görünene kadar, 30 ° C&#39;de 2-3 gün inkübe. Çıkan koloniler değerlendirmek ve kontrollerle karşılaştırmak. NOT: Genellikle büyük ve küçük koloniler elde edilir, ancak koloni boyutu ve hareket arasında bir korelasyonivity görülmektedir. Sekanslamadan taşınan yanlış pozitif en aza indirmek için bir 5-FOA sayacı seçimi tekniği (Adım 4) kullanılarak bu potansiyel hit Ekran. </li></ol> 4. 5-FOA ters seçim ve lekelenme Hatalı Tesbiti ortadan kaldırmak için <ol><li> Çift ve tek terk medya üzerine suret tek koloniler (örn SD-His-Ura ve SD-His plakaları) ve 30 ºC&#39;de gece boyunca büyümeye dışarı Streak. Vektör ve Hsp104 WT kontrolleri ile tekrarlayın. Üzerine kaplama SD-His önce 5-FOA bir 5-FOA üzerine konuldukları zaman hücreler, Hsp104 plazmid kaybetmiş ihtimalini artırarak 5-FOA aşamasının verimini artırır. </li><li> 5-FOA ekran yaparken kurumasını plakaları önlemek için, 4 ºC&#39;de SD-His-Ura parafılm plakaların ve mağaza sarın. Bu plakalar, sonuçta, sıralamadan için kullanılacaktır. </li><li> Streak gelen koloniler SD-His 5-FOA plakaları üzerinde tek koloniler plakaları (YPD medya + 1 g / L 5-FOA) ve 30 1-2 gün inkübe186. C tek koloniler görünür kadar. Burada, 5-FOA, URA3 genini ihtiva eden hücrelerden bir toksik ürün (5-florodeoksiüridin) dönüştürülür. Böylece, hala Hsp104 plazmid herhangi hücreleri ölür. </li><li> Şerit, SD-Ura ve SD-His plakaları üzerine iki kez 5-FOA plakalardan tek koloniler. Her Testi 3 koloni Her hit için en az bir koloni Hsp104 plazmid yitirmiş olacağı olasılığını artırmak çarptı. 30 ° C&#39;de 1-2 gün süreyle inkübe edilir. SD-His plakaları değil SD-Ura plakalar üzerinde büyüyecek Hsp104 plazmid kaybettik Kolonileri. </li><li> Deneyleri tespit için Hsp104 plazmidler kaybetmiş suşları büyür. Çalkalama ile 30 ºC &#39;de gecede 96 derin kuyu, 2 ml plakaları (örneğin sraff-His) rafınoz bırakma medyada doyurma suşları büyür. 5-FOA ile tedavi edilen kontrol suşları dahil emin olun. </li><li> Tahlil tespit için tabak hazırlayın. Bir çok kanallı pipet kullanarak, kısım 200 ul rafınoz bırakma medya (örneğin sraff-His) başına96 oyuklu bir plakanın her bir kuyucuğu, ayırma sütunları saf kültürlerin 1 ve 7. Kısım 250 kolon 1 içinde 16 doymuş kültürlerin her ul ve plakanın 7. </li><li> Seri olarak çok kanallı bir pipet ile iyice karıştırılması, plakanın her sütuna Kültürler 5 kat seyreltilir. Her çukurdaki nihai hacmi sağlamak üzere sütun 6 ve 12 ile seyreltilmiş kültür 50 ul çıkarın 200 ul&#39;dir. Bu bile lekelenme sağlamak esastır. </li><li> 96-cıvata çoğaltıcı aracı (frogger) kullanılarak, SD-His ve SGAL-His plakalar üzerine nüsha halinde kültürleri nokta. 2-3 gün boyunca 30 ºC de inkübe. </li><li> Kontrollere benzer SGAL-His tabakalarına toksisite sergileyen suşları dizilenmesi için seçin. Kontroller gibi toksik değil gibi yanlış pozitif suşları atın. 5-FOA ayrıca kendi sık sık toksik, yani tek başına hastalık alt tabaka daha SD-His plakalar üzerinde bir büyüme kusur veya bu sergi önemli ölçüde daha yüksek toksisite gösterirler suşları (atın <stronG&gt; Şekil 3). </li></ol> 5. koloni PCR ile Hsp104 değişkenleri sequencing <ol><li> Pençe ~ 10 ul PCR şeridi tüplerinde 3 ul 20 mM NaOH içinde SD-His-Ura plakalardan maya ve dondurulup eritilerek hücreler lize edildi. Daha sonra 10 dakika boyunca bir PCR 99 ° C&#39;de inkübe edilir, 10 dakika süre ile ya da sıvı azot içinde bir -80 ° C dondurucu içinde şerit tüpler yerleştirin. PCR sınıf su ile 100 ul suşları sulandırmak. </li><li> PCR reaksiyonları hazırlayın: 5 ul PCR şablonu, 0.5 ul 100 uM her bir primer, 0.5 ul 10 mM dNTP, PCR tamponu içinde 0.25 ul DNA polimeraz, ve PCR dereceli su ile bir 25 ul toplam hacme tamamlanır. Tasarım primerler rastlantısal bölgeyi artı iki uçta da yaklaşık olarak 100 bp amplifiye etmek. Başarılı amplifikasyon olasılığını artırmak için güçlendirilmiş bölge boyutunu en aza indirmek. Standart bir PCR programı çalıştırın. </li><li> Uygun sIz bir PCR ürünü amplifikasyon teyit etmek için agaroz jel elektroforezi ile analiz numuneleriör. Hiçbir ürün varsa PCR tekrarlayın. Not: PCR, yetmezliği genellikle aşama 5.1&#39;de kullanılan çok fazla maya kaynaklanmaktadır. </li><li> DNA sekanslaması için numune hazırlayın. Böyle Ekzonükleaz I ve Karides Alkalin Fosfataz enzim olarak PCR saflaştırma veya bir PCR ürünü temizleme ajanı kullanılarak ya PCR primerleri Kaldır (ExoSAP-IT) tek iplikli DNA&#39;yı için. Bu reaktif enzimatik daha yüksek verim sağlayan, tüzel kişiliği olmayan astar ve dNTPs düşürür. </li><li> PCR ürünleri dizisi. Iyice mutasyonları için sıralama kromotogramlarını analiz emin olun. Genellikle birden plazmidleri barındıran maya gibi mutasyonlar, belirli bir site (Şekil 4), iki nükleotid bir karışımı olarak gözlemlenebilir. </li></ol>

<ol>
	<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.

Burada maya proteinopathy modellerini kullanarak hastalıkla ilişkili alt tabakaların toksisitesi bastırmak potansiyelize Hsp104 varyantları izole bizim yaklaşım sunmak. Bu yaklaşımı kullanarak, varyantlarının büyük kütüphanelerin, tek sınırlandırma, 5-FOA İkinci ekrana geçmek varyantları sayı olmak üzere, yüksek bir verimlilik de taranabilir. 96 kuyu formatında bu adımları gerçekleştirerek, rutin 1-2 hafta boyunca 5-FOA aşamasında bir anda 200 hit kadar ekran. Ilk tarama aşaması esnası...

Maya proteinopathy modeller amiyotrofik lateral skleroz (ALS) ve Parkinson hastalığı (PD) 1-3 içeren protein-yanlış katlanması bozuklukları için geliştirilmiştir. ALS hastalarında misfold protein TDP-43 ve FUS sentezlenmesi, maya 1,2 sitoplazmik agregatlar oluşturması için zehirli ve mislocalize bulunmaktadır. Benzer şekilde, PD karıştığı α-sinüklein (α-sin), ekspresyonu, toksik ve mislocalizes maya 3 sitoplazmik eden agregalar meydana getirir. Bu özellikler bu bozuklukların 4,5 olan hastalarda fenotipleri özetlemek. Bu nedenle, maya modelleri önlemek ya da bu fenotipleri 2,6-13 ters proteinler ya da küçük moleküllerin taranmasına yönelik yararlı bir platform sağlar. Biz nedeniyle TDP-43, FUS ve α-syn için toplanmasını ve toksisite geri yeteneğine sahip proteinlerin geliştirilmesinde ilgilendi. Biz Hsp104, hem fr proteinleri ayırmak olma eşsiz yeteneği olan mayadan AAA + protein odaklanmakmayada om amorf agrega ve amiloid, henüz hiçbir insan homolojunu 14,15 sahiptir. Hsp104 ince endojen maya prionları parçalamak için ayarlanmış ve normalde 16,17 karşılaştığında asla insan nörodejeneratif hastalıklarda rol yüzeylerde, parçalamak için sadece sınırlı bir yeteneği vardır. Böylece, biz birebir bu insan substratları ayrıştırmak mümkün Hsp104 gelişmiş versiyonlarını mühendisi hedefliyoruz. Hsp104 hata eğilimli PCR kullanarak varyantları Bunu yapmak için, biz büyük kütüphaneler inşa; bu kütüphaneler maya proteinopathy modelleri 17 kullanılarak taranabilir. Hsp104 17 çok büyük olduğu gibi biz, kütüphaneler oluşturmak ve tarama için bir etki alanı hedefli bir yaklaşım benimsemiştir. Benzer yaklaşımlar diğer etki taranması için kullanılabilecek olsa biz başlangıçta, orta etki Hsp104 17 (MD) odaklanmıştır. Bu tür geri kalanı yüzey gösterimi gibi alternatif teknikler karşıt olarak bu modeller, doğrudan Disaggregase aktivitesi için elenmesini sağlamak18 bağlama izlenmesi için kullanılacak ricted. Bizim protokol iki tarama aşaması (Şekil 1) dayanmaktadır. İlk olarak, mayada hastalık substratın toksisitesi bastırmak Hsp104 varyantları seçilir. Bunu yapmak için, Hsp104 varyantları ve hastalıkla ilişkili alt-tabaka Δ hsp104 maya içine ortak transforme edilir. Biz, vahşi tip yokluğunda (WT) Hsp104 17 Hsp104 dizisi alan araştırmak Δ hsp104 maya kullanılmaktadır. Önemlisi, Hsp104 silinmesi mayada α-sin, FUs, ya da TDP-43 toksisite etkilemez, ve Hsp104 WT ifadesi minimum kurtarma 1,13,17 sağlar. Maya her iki proteinlerin sentezlenmesinin uyarılması için ortam uyarılması üzerine yerleştirilir. Koloninin hastalıkla ilişkili substrat bahşedebilir büyüme toksisitesi bastırmak Hsp104 varyantlarını barındıran maya. Koloniler toksisite kalıbı bastıramaz varyantlarını korurken, bu varyantlar, daha fazla analiz için seçilmiştir. Bununla birlikte,yanlış pozitif Bu ekranda önemli bir sorun vardır. TDP-43, FUS ve α-syn sentezlenmesi ifade edilir Hsp104 varyant olmayan toksisite kendiliğinden genetik bastırma ortaya çıkması için güçlü bir seçici basınç oluşturur, oldukça zehirlidir. Bu nedenle, biz ayrıca spesifik olmayan toksisite bastırıcılar 17 ortadan kaldırmak için, nispeten yüksek miktarda olan ikincil bir ekran kullanmaktadır. Bu ikincil bir tarama, seçilen maya Hsp104 plazmidin 19 seçme karşı 5-Fluorootic asit (5-FOA) ile muamele edilir. Soy daha sonra alt-tabakanın toksisite Hsp104 plazmidin kaybından sonra geri sağlamak için tahlil lekelenme ile (TDP-43, FUS ya da α-sin) toksisite alt-tabaka açısından değerlendirilir. Bu nedenle, toksisitesi bu ikinci ekranda geri edildiği maya muhtemelen başlangıçta bağlı Hsp104 varyantın varlığını toksisite bastırma gösterilir. Bu maya &quot;sonuç&quot; olarak adlandırılan ve Hsp104 plazmid daha sonra olmalıdırgeri kazanılmış ve Hsp104 gen 17 (Şekil 1) &#39;de başkalaşımların teşhis edilmesi için dizilenmiştir. Herhangi bir hitleri daha sonra toksisite bastırılması için yeniden test daha sonra bağımsız bir şekilde mutasyona inşa yer yönlendirmeli mutajenezi kullanarak ve teyit edilmelidir. Bu protokol için potansiyel uygulamalar geniştir. Bu yöntemler kullanılarak, protein herhangi bir tür kütüphaneleri maya içinde toksik olan herhangi bir alt tabaka proteininin toksisitesi bastırmak varyantlar için taranabilir olabilir.

<table border="0" cellpadding="0" cellspacing="0" width="766">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:320px;">Table of Specific Materials/Equipment</td>
			<td style="width:227px;"></td>
			<td style="width:132px;"></td>
			<td style="width:87px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GeneMorphII EZClone Domain Mutagenesis Kit</td>
			<td>Agilent</td>
			<td>200552</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">150mm Petri dishes</td>
			<td>Falcon</td>
			<td>351058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Fluorootic Acid</td>
			<td>Research Products International</td>
			<td>f10501-5.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-DeepWell 2mL Plates</td>
			<td>Eppendorf</td>
			<td>0030 502.302</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 bold replicator tool</td>
			<td>V&#38;P Scientific</td>
			<td>vp-404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ExoSAP-IT</td>
			<td>Affymetrix</td>
			<td>78200</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating potentiated hsp104 variants using yeast proteinopathy models

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

Biz orta etki randomize Hsp104 varyantlarının bir kütüphanesi inşa TDP-43 toksisite bastırılması için gösterildi. Kütüphane dönüştürülmüş ve ekranın darlığı değerlendirmek için glukoz, galaktoz levhaları (Şekil 2) üzerine kaplanmıştır. Tek tek koloniler seçilir ve suşlar karşı Hsp104 plazmid ortadan kaldırmak için 5-FOA kullanılarak seçilmiştir. Bu suşlar o toksisite onaylamak için değerlendirildi Hsp104 varyantları olmadan, TDP-43 tek başına nedeniyle oldu....

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.

Kuvvetlendiği Hsp104 Varyantlar kullanarak Maya Proteinopathy Modelleri Ayırma

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

Video: Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

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

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

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

Nicel Değerlendirme Elektrosprey İyonizasyon Kütle Spektrometresi kullanarak Maya Lipidome

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

Biyoloji

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.

İnsan DNA Onarım Proteinler Genetik Araştırmalar Maya Model Sistemi olarak kullanma

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.

Maya Colony Gömme Yöntemi

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.

Yeni Cerrah Kemirgen Cerrahisi İlkeleri

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 Tutucu ve Fare ve Sıçanlarda Ortak Bileşik İdaresi Rotalar

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.

Yüksek verim Maya Plazmid overekspresyonu Ekran

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.

Hsp104 saflaştırılması, 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 ...

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.

I · Cryo Kit kullanarak Fare Sperm Kriyoprezervasyon ve Kurtarma

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.

SPLINTER kullanarak Pooled Sıralama gelen Nadir Genomik Variantların Algılama

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.

Elektroforetik Hareketlilik Assay (EMSA) ve Shift kullanarak Fonksiyonel Olmayan kodlama Genetik Türevleri için tarama DNA afinite Yağış Deneyi (DAPA)

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