Bu çalışma NIAID (R01AI074951 U54AI057168) ve SC Penn Genom Frontiers Enstitüsü hibeleri ile desteklenen; NIH T32HG000046 TSM; NIH T32GM07229 ve T32AI007324 LRS.

Bölüm 1: 384 Şey Tabaklar RNAi <ol><li> Tarama için kullanılan reaktifler Standardizasyon esastır. Biz bireysel birçok Schneider medya, fetal sığır serumu, ve boyama reaktifleri test edin. Sonra biz, tüm ekranı için aynı çok kısım ve kullanın. </li><li> Herhangi bir tahlil için, salt biyolojik en müsait olduğunu belirlemek için birkaç farklı hücre hatları test etmek en iyisidir. Biz genellikle S2 DRSC hat kullanın. </li><li> 25 ° C tam Schneider medya Drosophila hücreleri büyütün. <ol class="lalpha"><li> Schneider medya </li><li> % 10 ısı inaktive FBS </li><li> 1X L-glutamin </li><li> 1X Kalem / strep antibiyotik </li></ol></li><li> Drosophila hücreleri yarı yapışık ve her dört günde bir, matara kapalı hücreleri pipetleme ve taze bir balona 01:10 hücreleri seyreltilmesi trypsinization olmadan geçişli. Hücreler deneyler için log fazı olmalıdır. </li><li> Plaka-plaka ve günden güne değişkenlik tüm sıvı eklemeleri için çok iyi plakaları otomatik sıvı taşıma kullanımı ile en aza indirilmiştir. Biz WellMate (Matrix) kullanın. </li><li> WellMate tüp hazırlayın. <ol class="lalpha"><li> Sprayhood ve% 70 etanol ile WellMate. </li><li> % 70 etanol ile ambalaj ve sprey tüp çıkarın. </li><li> WellMate pozisyon dağıtım boru kaset takın. </li><li> 25 ml% 70 etanol ile Başbakan boru. </li><li> 15 dakika, sonra başbakan başka bir 25 mL etanol etanol içinde tüp sterilize edin. </li><li> 50 ml steril PBS ile astarlanırsa boru durulayın. </li></ol></li><li> Şişeyi ve sayısı hücreleri yerinden oynatın. Pelet hücreleri (300xg, 5 dk)% 25 daha fazla olduğunu. Ortalama numaralı seribaşı hücrelerinin sayısı tahlil uzunluğuna göre optimize edilmesi gerekir. Otomatik görüntü analizi yaparken, hiçbir hücre topaklanma ile tek tek tabakalı bir görüntü segmentasyonu için idealdir. </li><li> 1.7x10 6 hücre / ml serum serbest Schneider medya pelet hücreleri tekrar </li><li> Biz 384 kuyucuğu 250ng/well bir konsantrasyonda dsRNA piyasada mevcut kütüphanesi ile önceden aliquoted ekran gerçekleştirmek. Her plaka birkaç kuyu elle eklenir, kontroller için boş bırakılır. Biz bir negatif kontrol olarak lusiferaz dsRNA kullanın. Biz güçlü bir RNAi kontrol olarak karşı dsRNA Konu (dIAP) kullanın. Dramatik hücre ölümü bu anti-apoptotik faktör sonuçlarının yıkmak ve aşağı knock kalitesini değerlendirmek için hızlı bir görsel yoludur. Son olarak, biz, bizim tahlil okuma kullanarak viral enfeksiyon inhibe doğrulamak için bir pozitif kontrol olarak, bu durumda beta-galacosidase viral muhabiri karşı dsRNA kullanabilirsiniz. Hücre tohumlama, kısım 1 ul dsRNA kolay görünüm için izin vermek için iyi köşeye zamanda plaka dsRNA lekelenme, daha sonra hücrelerin eklemeden önce kuyunun altındaki (300xg, 1 dak.) Üzerine çift iplikçikli santrifüj. </li><li> Her bir kuyu için yukarıda hazırlanan 10 mcL hücre eklemek için WellMate kullanın. </li><li> 300xg, 1 dakika santrifüj plakalar üzerine hücreleri Spin. </li><li> 45 dakika boyunca 25 ° C inkübatörde inkübe edin. </li><li> 20 mcL tam Schneider medya ekleyin ve 300xg, 1 dakika santrifüj. </li><li> Nemlendirilmiş Tupperware kapları koyun tabak, suda ıslatılmış kağıt havlu ile kaplı. 384 hacmi küçük, böylece, buharlaşma da kenarına kuyuların okumaları artefakt değişikliklere yol açabilir biyoloji, büyük etkileri olabilir. Bunu aşmak için, tabak, yüksek nem altında tutulmalıdır. </li><li> Sağlam demonte izin vermek için üç gün boyunca inkübe edin. Ortamı değiştirmek veya bu süre içinde nemlendirilmiş kapları açmak için gerek yoktur. </li></ol> Bölüm 2: Vaccinia Virüs ile Enfekte <ol><li> 15 dakika boyunca Quatricide sterilize edilen çok-kanallı aspiratör, daha sonra% 70 etanol içinde durulayın. </li><li> Yukarıda açıklandığı gibi WellMate hazırlayın. </li><li> Serum serbest medya tam bir medya 01:05 seyreltilmesi ile enfeksiyon için 2% FBS ile Schneider medya hazırlayın. </li><li> 0,8 um şırınga aracılığıyla hücresel enkaz kaldırmak veya saflaştırılmış virüs kullanmak için filtre Filtre ham virüs stokunun. </li><li> 2 enfeksiyon çokluğu (İçişleri Bakanlığı) elde etmek için% 2 serum Schneider medya vaccinia virüs sulandırınız. </li><li> WellMate boru PBS çıkarın ve tüp hava kabarcıkları serbest kadar seyreltilmiş virüs başbakan. </li><li> DsRNA ile tedavi edilen Drosophila hücreleri plakaları medya hafifçe kaldırmak için sterilize çok kanallı aspiratör ve vakum manifoldunun kullanın. </li><li> WellMate kullanarak plakaları üzerine de başına 30 mcL virüs koyun. </li><li> 300xg plakaları 1 dakika santrifüjleyin. </li><li> WellMate boru sterilize edin. 25 mL% 10 blea ile Başbakanch, 10 dakika bekleyin ve bir 25 ml% 10&#39;luk çamaşır suyu ile başbakan. </li><li> Başbakan tüp 50 ml su ile 50 ml% 70 etanol ile izledi. Ambalajın boru dönün. </li><li> Nemlendirilmiş Tupperware konteyner plakaları geri dön. 48 saat boyunca 25 ° C&#39;de inkübe edin. </li></ol> Bölüm 3: Boyama Tabaklar <ol><li> 10 dakika için 15 mcL% 4 formaldehit / PBS plakaları sabitleyin. Sıvı bir multi-kanal aspiratör ve vakum manifoldunun her adımı kullanarak kaldırılır. Plakalar, steril ekipman giderildi var ve reaktifler artık gerekli değildir. </li><li> Kaldır düzeltmek ve 30 mcL PBST (PBS +% 0,1 Triton-X) eklemek için kullanabilirsiniz WellMate. Ve tekrar 10 dakika inkübe edin. Tüp sterilize edilmelidir gerekmez hariç İşlet, yukarıda açıklandığı gibi WellMate. </li><li> Vaccinia enfeksiyonu saptamak için erken / geç vaccinia organizatörü tarafından yönlendirilen bir β-galaktosidaz muhabiri kullanın. </li><li> 10 dakika boyunca sıvı ve inkübe blok hücreleri (2% BSA ile PBST) çıkarın. </li><li> Blok primer antikor sulandırınız, sıvı kaldırmak ve her biri de çok kanallı kullanarak tekrar pipet (Matrix) 15 mcL antikor ekleyin. </li><li> Levhalar (300xg, 1 dak) Spin, net bir etiket ile kaplayın ve 4 ° C gecede inkübe. </li><li> Primer antikor çıkarın ve PBST 3 kez, her biri 10 dakika kuyuların yıkamak için WellMate kullanın. </li><li> Floresan ile işaretlenmiş sekonder antikor (FITC) ve blok içinde leke nükleer seyreltin ve multi-kanal tekrar pipet kullanarak kuyulara 15 mcL ekleyin. </li><li> Inkübe plakalar 1 saat oda sıcaklığında, ışıktan korunmalıdır. </li><li> Kuyu PBST 3 kez, WellMate kullanarak her biri 10 dakika yıkayın. </li><li> 4 net etiket, kapak ve mağaza ile plaka Seal ° C üç hafta kadar. </li></ol> Bölüm 4: Görüntüleme ve Görüntü Analiz <ol><li> 20X büyütmede otomatik bir mikroskop görüntü tabak kullanın. </li><li> Toplam çekirdekleri (DAPI) ve enfekte olmuş hücreleri (FITC) görüntüleri yakalayın. Dinamik aralık maksimize etmek için, her kanal için ayrı ayrı pozlama süresi optimize edin. </li><li> Beklenen odak düzlemi altındaki ve üstündeki uçakları yayılan görüntüleri, arayla 1um, Z yığını alarak otomatik odaklama fonksiyonu ince ayar yapın. Optimal odak düzlemli bulunmaktadır, buna göre otomatik odaklama ayarlarını. </li><li> Resim tüm plaka, Hoechst kanal (DAPI) ve virüs kanal (FITC) başına en az üç fotoğraf çekmek. Bir bütün olarak iyi temsil eden görüntüler yakalamak için iyi bir içinde birkaç farklı bölgelerden gelen siteleri seçin. </li><li> Segment resmi bir dergide yazmak ve her plaka için nükleer boyanma (toplam hücreleri) ve enfeksiyon (FITC) pozitif hücrelerin toplam sayısını hesaplamak için Kont Çekirdekler modülleri kullanmak için yazılım MetaXpress kullanın. </li><li> Yüzde enfeksiyon (100 * FITC pozitif / toplam hücre) hesaplayın ve dönüşümü giriş. </li><li> Adayları belirlemek için, her plaka medyan ve çeyrekler açıklığı dayalı sağlam bir Z skoru kullanın. Bu yöntem aykırı ve nonsymmetric veri duyarsız ve böylece, hatta zayıf veya orta derecede hit 10 tanımlanması daha fazla güç sağlar. Her plaka için, medyan log-dönüştürülmüş verileri çıkarılır. Daha sonra elde edilen değerler 0.74 kez çeyrekler açıklığı (75 persentil ve 25. yüzdelik değerleri arasındaki fark) tarafından ayrılır. 0,74 faktörü yaklaşık bir standart normal dağılım için kullanılır. </li><li> Sağlam Z skoru plaka medyan standart sapma mesafe bir ölçüsüdür. Örneğin, 2 sağlam bir Z-skoru iyi% enfeksiyonu plaka medyan veya p &lt;0.05 ~ 2 standart sapma gösterir. </li><li> Hücre sayısı, sitotoksik adayları belirlemek için analiz edin. Nükleer sayar sağlam Z skoru hesaplayın. Adaylar ile sağlam bir yinelenen ekranlarında Z &lt;-2 sitotoksik olarak kabul edilir. </li><li> Sitotoksisite olmadan&gt; yinelenen ekranlarında 2 veya &lt;-2 (p &lt;0.0025) yüzde enfeksiyon için sağlam bir Z skoru Wells birincil ekran potansiyel aday olarak kabul edilir. </li><li> Olumlu adayların bağımsız reaktifler kullanılarak doğrulanır. dsRNAs mRNA başka bir bölgeye ilgi genlerin karşı oluşturulan ve yukarıdaki gibi test edilmektedir. </li><li> Iki bağımsız dsRNAs aynı fenotipe sahip olduğunuz bu genlerin daha fazla çalışma için kabul edilebilir. </li></ol> Bölüm 5: Temsilci Görüntüler ve Enfeksiyon Oranları yorumlanması <img src="/files/ftp_upload/2137/2137fig1.jpg" alt="Şekil 1" /> Şekil 1 bulaşmamış kuyu iyi bulaşmış bir temsilcisi immünofloresan mikroskobu ile ölçülen vaccinia ifade beta-galaktosidaz (βgal) protein, pozitif boyanma hücreleri içeren, vaccinia virüs proteinler için herhangi bir boyama görünmüyor.Virüs = yeşil, çekirdeklerin = mavi. <img src="/files/ftp_upload/2137/2137fig2.jpg" alt="Şekil 2" /> Şekil 2 Otomatik görüntü analiz yazılımı, kullanıcı tarafından belirlenen parametreler kullanılarak her görüntü enfeksiyon rakamlarla . Bir temsili görüntü, hücre çekirdekleri ve viral antijen eksprese eden hücrelerin sayısı toplam sayısı, büyüklüğü ve yerel arka plan boyanma üzerinde boyanma yoğunluğu dayanan sayılır. <img src="/files/ftp_upload/2137/2137fig3.jpg" alt="Şekil 3" /> Şekil 3 iplik (dIAP) demonte hedef genlerin sağlam RNAi tükenmesi için bir pozitif kontrol olarak hizmet vermektedir. Bu anti-apoptotik faktör demonte dramatik hücre ölümüne yol açar. Çekirdekler = mavi. <img src="/files/ftp_upload/2137/2137fig4.jpg" alt="Şekil 4" /> Şekil 4 lusiferaz demonte enfeksiyon çift iplikli RNA tedavi etkisi için bir negatif kontrol olarak hizmet vermektedir. Lusiferaz tükenmesi, tedavi edilmezse hücrelere göre enfeksiyon üzerinde hiçbir etkisi yoktur. Virüs = yeşil, çekirdeklerin = mavi. <img src="/files/ftp_upload/2137/2137fig5.jpg" alt="Şekil 5" /> Şekil 5 βgal protein demonte enfeksiyon okundu olarak tahlil βgal protein düzeyleri kullandığından beri, bir enfeksiyon azaltılması için bir pozitif kontrol olarak hizmet vermektedir. Enfekte hücrelerin yüzdesinde bir azalma βgal sonuçlar tükenmesi. Virüs = yeşil, çekirdeklerin = mavi. <img src="/files/ftp_upload/2137/2137fig6.jpg" alt="Şekil 6" /> Şekil 6, enfekte hücrelerin yüzdesinde bir azalma hücresel faktör Rab5 sonuç demonte . Rab5 endositoz katılmak için bilindiğinden, bu faktör, büyük olasılıkla vaccinia virüs giriş katkıda bulunmaktadır. Bu ekran bir örnek uç temsil eder. Virüs = yeşil, çekirdeklerin = mavi.

<ol>
	<li>Ramadan, N., Flockhart, I., Booker, M., Perrimon, N., Mathey-Prevot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+high-throughput+RNAi+screens+in+cultured+Drosophila+cells.">Design and implementation of high-throughput RNAi screens in cultured Drosophila cells.</a> Nat Protoc. 2, 2245-2264 (2007).</li><li>Chotkowski, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=West+Nile+virus+infection+of+Drosophila+melanogaster+induces+a+protective+RNAi+response.">West Nile virus infection of Drosophila melanogaster induces a protective RNAi response.</a> Virology. 377, 197-206 (2008).</li><li>Blondel, D., Petitjean, A. M., Dezelee, S., Wyers, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+stomatitis+virus+in+Drosophila+melanogaster+cells:+regulation+of+viral+transcription+and+replication.">Vesicular stomatitis virus in Drosophila melanogaster cells: regulation of viral transcription and replication.</a> J Virol. 62, 277-284 (1988).</li><li>Sessions, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+insect+and+human+dengue+virus+host+factors.">Discovery of insect and human dengue virus host factors.</a> Nature. 458, 1047-1050 (2009).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+RNAi+screening+in+Drosophila+S2+cells:+what+have+we+learned+about+host-pathogen+interactions?.">Genomic RNAi screening in Drosophila S2 cells: what have we learned about host-pathogen interactions?.</a> Curr Opin Microbiol. 11, 262-270 (2008).</li><li>Hao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+RNAi+screen+identifies+host+genes+important+for+influenza+virus+replication.">Drosophila RNAi screen identifies host genes important for influenza virus replication.</a> Nature. 454, 890-893 (2008).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPI+activity+coupled+with+fatty+acid+biosynthesis+is+required+for+viral+replication.">COPI activity coupled with fatty acid biosynthesis is required for viral replication.</a> PLoS Pathog. 2, e102-e102 (2006).</li><li>Cherry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screen+reveals+a+specific+sensitivity+of+IRES-containing+RNA+viruses+to+host+translation+inhibition.">Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition.</a> Genes Dev. 19, 445-452 (2005).</li><li>Moser, T. S., Jones, R. G., Thompson, C. B., Coyne, C. B., Cherry, 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+Kinome+RNAi+Screen+Identified+AMPK+as+Promoting+Poxvirus+Entry+through+the+Control+of+Actin+Dynamics.">A Kinome RNAi Screen Identified AMPK as Promoting Poxvirus Entry through the Control of Actin Dynamics.</a> PLoS Pathog. 6, e1000954-e1000954 (2010).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. 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+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4, 67-73 (1999).</li><li>Echeverri, C. J., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+RNAi+screening+in+cultured+cells:+a+user's+guide.">High-throughput RNAi screening in cultured cells: a user's guide.</a> Nat Rev Genet. 7, 373-384 (2006).</li></ol>

Drosophila genom RNAi tarama viral enfeksiyon hücresel bileşeni incelenmesi için sağlam ve etkili bir yöntem sağlar. Ev sahibi içsel immün yanıt bileşenleri ve terapötik hedefleri temsil edebilir viral replikasyon için gerekli ana protein faktörleri tanımlamak için kullanılır. Drosophila hücreleri, bir çok memeli virüs bulaştırmak. Bir ekran, tahlil yapmadan önce dikkatli bir hücre tipi, hücre sayısı ve enfeksiyon seviyesi için optimize edilmiş olmalı ve 11 pozitif ve negatif viral ve hücresel hedefleri de dahil olmak üzere kontrol edilmelidir. Önce tarama testinin dinamik aralığı en üst düzeye çıkarmak için pozitif ve negatif kontroller arasında yeterli ayırma sağlamak için önemlidir. Ekran yapıldıktan sonra, adaylar, ev sahibi mekanizmaları enfeksiyona katkıda bulunan belirlemek için işlevsel kategoriler halinde bölünmüş olabilir. Vaccinia gibi memeli virüsler için, RNAi memeli homologları katkısı ikincil analizi bir parçası olarak tespit edilmelidir. Birlikte ele alındığında, bu güçlü tarama yöntemlerinin bize virüsler, konak hücreleri ile etkileşimde bulunduğu karmaşık mekanizmaları anlamak için izin verir.

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<td>Cell culture</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Schneider&#x2019;s media</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11720</td>
<td>&nbsp;</td>
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<td>GlutaMAX 100X</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>35050</td>
<td>&nbsp;</td>
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<td>Penicillin/streptomycin (pen/strep)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS)</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>F6178</td>
<td>&nbsp;</td>
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<td>Screening</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Genome-wide dsRNA library</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>&nbsp;</td>
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<td>384 well plates, black with clear bottom</td>
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<td>Corning</td>
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<td>Clear seals</td>
<td>&nbsp;</td>
<td>Denville</td>
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<td>&nbsp;</td>
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<td>Well Mate</td>
<td>&nbsp;</td>
<td>Matrix</td>
<td>201-10001</td>
<td>&nbsp;</td>
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<td>24 pin manifold</td>
<td>&nbsp;</td>
<td>Drummond</td>
<td>3-000-101</td>
<td>&nbsp;</td>
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<td>Hoechst 33342</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B2261</td>
<td>&nbsp;</td>
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<td>Fluorescently-labeled secondary antibodies</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
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<td>ImageXpressMicro automated microscope</td>
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<td>MetaXpress image analysis software</td>
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<td>Molecular Devices</td>
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rnai screening for host factors involved in vaccinia virus infection using drosophila cells

Yalnızca virüs, insan hastalığının doğal salgın hastalıkların neden olabilir, ancak biyoterörizm onların potansiyel kullanımı da bir endişe, viral patojenlerin önemli bir kamu sağlığı tehdidi temsil eder. Hücresel faktörlerin daha iyi anlaşılması, çok ihtiyaç duyulan terapötikler etkisi enfeksiyon gelişimini kolaylaştıracağını söyledi. Çeşitli organizmaların tam genomu sıralama ile birleştiğinde RNA enterferans (RNAi) teknolojisi son gelişmeler genom, hücre tabanlı kayıp fonksiyon-ekranlar optimizasyonu yol açmıştır. Drosophila hücreleri nedeniyle genom ölçekli ekranlar özellikle mükellef bu sistemi 1 RNAi kolaylığı ve verimliliği. Önemlisi, virüslerin Drosophila hücreleri, tıbbi ve tarımsal önemi 2,3,4 memeli virüslerin sayısı da dahil olmak üzere geniş bir yelpazede bulaşabilir. Drosophila Önceki RNAi ekranları giriş, çeviri ve RNA çoğaltma 5 virüs enfeksiyonu da dahil olmak üzere çeşitli adımlar için gerekli konakçı faktörleri tespit ettik. Ayrıca, Drosophila hücre kültürü viral replikasyon için gerekli olan hücresel faktörlerin birçoğu da bu virüsler 4,6,7,8, 9 ile enfekte insan hücrelerinde sınırlayıcı. Bu nedenle, viral enfeksiyon sırasında eş-tarafından tercih ana faktörlerin belirlenmesi, antiviral tedavi için yeni hedeflere sunar. Burada bir örnek olarak vaccinia virüs kullanarak, viral enfeksiyon yer hücresel faktörleri belirlemek için bir yüksek verim RNAi ekran için genelleştirilmiş bir protokol mevcut.

Watch this Scientific Journal Video about RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells at JoVE.com

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral enfeksiyon katılan Roman konakçı faktörleri fonksiyonu RNAi tarama hücre tabanlı genom zarara tespit edilebilir. A<em&gt; Drosophila</em&gt; Hücre kültürü modeli RNAi kolaylığı ve verimliliği nedeniyle bu yaklaşım, özellikle mükellef. İşte biz bu tekniği kullanarak göstermek<em&gt; Vaccinia</em&gt; Örnek olarak virüs.

RNAi Tarama için ilgilendim Ana Faktörler Vaccinia Virüs Enfeksiyonu Drosophila Hücre

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

rnai-screening-for-host-factors-involved-vaccinia-virus-infection

Research

JoVE Journal

Immunology and Infection

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

9.4K Görüntüleme.  University of Pennsylvania. Viral enfeksiyon katılan Roman konakçı faktörleri fonksiyonu RNAi tarama hücre tabanlı genom zarara tespit edilebilir. A Drosophila Hücre kültürü modeli RNAi kolaylığı ve verimliliği nedeniyle bu yaklaşım, özellikle mükellef. İşte biz bu tekniği kullanarak göstermek Vaccinia Örnek olarak virüs.

Video: RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

Model Herpesvirüs Litik çoğaltma Diseksiyon Host-virüs Etkileşim

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

İmmünoloji ve Enfeksiyon

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

Genler belirlenmesi için VIGS-aracılığı ile İleri Genetik Tarama konakçı olmayan Direnç ilgilendim


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Floresan Etiketli Vaksinia Virüs Proteinlerinin Verimli Üretimi için Metodoloji

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Analizi<em&gt; Yersinia enterocolitica</emAna Hücreleri içine&gt; Efektör Translokasyon Beta-laktamaz Effector Fusions kullanma

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Basit ve Etkin Yaklaşım Mutant Vaccinia Virüs Vektörler Construct

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Yer Alan Ana Faktörler yüksek verimli tarama mikroskopisi bazlı deneyler<em&gt; Brucella</em&gt; Hela Hücrelerinin Enfeksiyon

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

Deneyleri roman endotel düzenleyiciler inflamatuvar yanıtta yer alan karakterize etmek için eleme

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

Araçlar Viral Immunomodulators ve ana bilgisayar Antiviral faktörler tanımlanması için eleme olarak Abdovirüs enfeksiyonlar

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

İki fotonlu Intravital mikroskobu kullanılarak grip virüsü enfeksiyonu sırasında trakeal mukozasında görüntüleme hücre etkileşimi

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

RNAi Tarama için ilgilendim Ana Faktörler Vaccinia Virüs Enfeksiyonu Drosophila Hücre

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