Name: evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy
Uploaded: 2016-09-05T14:00:00.000+00:00
Duration: 12 min 3 s
Description: Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

Burada sunulan çalışma Sağlık Araştırması (CIHR) Kanada Enstitüleri tarafından desteklenmiştir (DCB-120266, PPP-133377 ve AG için HBF-348967 verir).

1. Hücre ve Transfeksiyonlar <ol><li> Dulbecco&#39;nun modifiye Eagle ortamında (DMEM) içinde kültür HEK 293T hücreleri,% 10 cenin sığır serumu (FBS) ve% 1 penisilin / streptomisin ile takviye edilmiştir. Hücre kültürü ortamı içinde 2 x 10 5 hücre / ml&#39;lik bir süspansiyon hazırlayın. Her çukuruna hücre süspansiyonundan 500, 100 ve 1000 ul, 24 oyuklu, 96 gözlü, 12 gözlü levhalar, viral üretimi, hücre canlılığı ve immün aktivasyon deneyleri sırasıyla (Şekil 2A) için. </li><li> Yavaşça girdap plakalar ve% 5 CO 2 ile 37 ° C&#39;de / Ç N onları kuluçkaya yatmaktadır. % 50-70 confluency hücreleri büyür. </li><li> Bir transfeksiyon planına göre, 1.5 ml mikro tüpler (örneğin Şekil 2B) test RNA&#39;lar ve kontrol seyreltileri hazırlanır. <ol><li> Viral üretimi deneyi için, bir HIV-1 ifade plazmidinin 10 ng / | il seyreltme hazırlanması ve her bir tüpe 10 ul ekle. Sonraki 5 uM test RNA&#39;lar dilüsyonları ve negatif kontrol RN hazırlamakC. 25 ya da 100 nM nihai konsantrasyonlarda, ilgili tüplere her bir test RNA ve negatif kontrol seyreltme 2.5 ya da 10 ul ekle. </li><li> C: Hücre canlılığı deneyi için 5 uM test RNA dilüsyonları ve pozitif kontrol RNA, düşük molekül ağırlıklı poli I bir 10 mg / ml seyreltme hazırlar. sırasıyla, 100 nM veya 200 ug / ml nihai konsantrasyonları, tekabül eden tüpler test RNA ya da pozitif kontrol RNA dilüsyonları 2 ul ekle. </li><li> immün aktivasyon deneyi için, deney RNA veya adım 1.3.2 hazırlanan pozitif kontrol RNA dilüsyonları 20 ul ekle. sırasıyla, 100 nM veya 200 ug / ml nihai konsantrasyon, tekabül eden tüplere. Aşama 1.3.1 ng 25 ve 100 nihai miktarda vererek, test RNA ekspresyon plazmidleri, deney RNA&#39;lar 5 uM dilüsyonları yerine 10 ng / | il dilüsyonları hazırlamak: edin. 100 adım 1.3.2 ng. ve 1.3.3. </li></ol></li><li> Viral ürünler için, her transfeksiyon tüpüne DMEM 50, 25 ya da 75 ul eklesırasıyla uction, hücre canlılığı ve bağışıklık aktivasyon tahlilleri. Viral üretim deneyleri için, bir sonraki adımdan önce bir biyo-güvenlik seviyesi 3 (BSL3) laboratuvara hücreleri ve hazırlanan transfeksiyon tüpleri getirmek. </li><li> transfeksiyon tüplerine transfeksiyon reaktif sırayla 2 ul ekleyin ve kompleksleri oluşturmak için izin vermek için 15 ila 20 dakika inkübe edin. Belirli transfeksiyon reaktif Malzemesi Tabloya bakınız kullanılacak. </li><li> damla damla hücre kültür plakaları içinde karşılık gelen pozisyonlarda, her mikro tüp tamamını transfeksiyon karışımı ekleyin. Yavaşça girdap ve% 5 CO2 ile 37 ° C &#39;de 48 saat süre ile inkübe edin. </li><li> Hücre kültürü plakaları (Şekil 2C), HIV-1 üretimini (Bölüm 2), hücre canlılığı (bölüm 3) ve immün aktivasyon (bölüm 4) ölçün. </li></ol> 2. Viral Üretim Deneyi <ol><li> inkübatör 24-iyi hücre kültür plakaları çıkarın ve BSL-3 hücre kültürü içindeki girdap plakalar hafifçedavlumbaz. HIV-1 üretimini ölçmek için kullanılacak olan 96-gözenekli düz tabanlı plaka içinde de karşılık gelen her gözden üst fazın 150 ul aktarın. Not: Genel viral miktar deneyleri, enzim-bağlı immünosorbent deneyi (ELISA), 20, ters transkripsiyon polimeraz zincir reaksiyonu (RT-PCR) 21 yoluyla viral RNA miktarının HIV-1 kapsid proteini (p24) ekspresyonunu ölçmek ve aktivitesinin ölçülmesi bulunmaktadır HIV-1 RT enziminin. 2.2 2.5 HIV-1 RT aktivitesinin 13,15,16 ölçmek için yöntemler açıklar Adımları. </li><li> Aktarım, bir viral bozulması kokteyli 15 (Tablo 1) 25 ul ihtiva eden 96 oyuklu bir plaka gözenekleri içinde karşılık gelen süpernatan 5 ul. Oda sıcaklığında 5 dakika süreyle inkübe karışımı ve radyoaktivite iş istasyonuna plaka aktarın. Not: Adım 2.2. Bir radyoaktivite iş istasyonu BSL3 laboratuarda mevcut değilse gereklidir. Plakalar kaldırılması gerekmiyorsaBSL3 laboratuar, 2.3 adıma geçin. ve radyoaktif kokteyl 25 ul yerine viral / radyoaktif bozulması kokteyli (Tablo 1) 50 ul ekle. </li><li> Radyoaktif bir kokteyl 15 (Tablo 1) Hazırlama ve viral süpernatan ve baskıyı kokteyl her çukuruna 25 ul ekle. 2 saat boyunca 37 ° C&#39;de inkübe edin. </li><li> Nokta 5 cam elyaf Di Etil Amino Etil (DEAE) Filtermat kağıdı karşılık gelen kareler üzerine reaksiyon karışımının ul ve noktalar 10 dakika kurumasını bekleyin. hiçbir iki numune doğrudan birbirlerine yatılı böylece her meydanda reaksiyon karışımı nokta. Bu aşamada 2.6 dakikada (cpm) başına sayıları belirlerken örnekler arasında çapraz-over önlemeye yardımcı olur. </li><li> Kağıtlar% 95 etanol ile iki kez 1 dakika yıkama yapılmıştır 2x tuzlu su, sodyum sitrat (SSC) tampon maddesi (Tablo 1) 5 dakika için 5 kat yıkayın. kağıtlar kurumaya ve örnek torbalarına bunları mühürlemek için izin verir. </li><li> Klip örnek çanta coBir kaset içine Filtermat kağıt ntaining ve bir mikroplaka sintilasyon sayacı içine kaset takın. Deneyde kullanılan [32 P] dTTP parti için sağlanan referans tarihi ile 32 P cpm okumak için sayaç ayarlayın. plaka haritayı kullanarak okuma ve sayaç başlatmak için hangi örnekler seçin. </li><li> herhangi bir virüs ölçümü yöntemi için, komşu negatif kontrol ile her test RNA için elde edilen değerler bölmek ve test RNA&#39;ların her tekrarında HIV-1 üretiminin yüzde inhibisyonunu elde etmek için 100 ile bu değeri çarpın. Farklı konsantrasyonlarda farklı test RNA karşılaştırma sonuçlarının bir örneği Şekil 3&#39;te verilmektedir. </li></ol> 3. Hücre Canlılık Testi <ol><li> / Ml MTT (3- [4,5-dimetil-2-tiyazolil] -2,5-difenil-2H-tetrazolyum bromid) Dulbecco fosfat tamponlu tuzlu su içinde seyreltildi (inkübatör, 96 gözlü levhalar çıkarın ve 5 mg 20 ul her bir kuyunun DPBS). f inkübeveya 37 ° C&#39;de 3 saat. </li><li> deterjan (% 1 NP-40, izopropanol içinde 4 mM HCI) her ile asitleştirildi izopropanol 150 ul ilave edin ve oda sıcaklığında 2 saat boyunca inkübe edin. </li><li> Bir mikrolevha spektrofotometre 570 nm&#39;de absorbans belirler. </li><li> bitişik transfeksiyon kontrolü ile her numune için elde edilen değer bölünerek, her pozitif kontrol ve test RNA göreceli MTT metabolizmasını hesaplayın. Farklı test RNA ve pozitif kontrol karşılaştırma sonuçlarının bir örneği Şekil 4&#39;te verilmektedir. </li></ol> 4. Bağışıklık Aktivasyon Testi <ol><li> inkübatör 12-kuyu plakaları kaldırmak ve kültür ortamı aspire. Yavaşça DPBS hücreler iki kez yıkayın ve her bir (proteaz ve fosfataz inhibitörleri, Tablo 1 de dahil olmak üzere), soğuk lisiz tamponu 22 70 ul ekle. Buz üzerinde 10 dakika süreyle inkübe edin. </li><li> mikro tüpler hücre lizatları aktarın ve hızlı daldırarak bunları dondurmaksıvı nitrojen içinde borular. Numuneler Çözülme ve 3 olmak üzere toplam 2 daha donma çözülme döngüleri için tekrar izin verin. </li><li> Hücre yıkıntıları pelet haline getirildi, 4 ° C&#39;de, 15.700 x g&#39;de 15 dakika boyunca santrifüj lizatları. Yeni bir mikro tüpler süpernatantı aktarın ve Coomassie mavisi (Bradford) yöntemini 23,24 kullanarak protein konsantrasyonunu belirleyin. </li><li> % 10 denatüre edici poliakrilamid jel içinde her bir numunenin proteinin 75 ug çözmek ve daha önce 25,26 tarif edildiği gibi bir nitroselüloz zara transfer. </li><li> Bir membrana elektroforezi ve aktarımı sonra, iki kez distile su ile yıkama, ardından 1 dakika boyunca Ponceau S (Tablo 1) &#39;de membran inkübe edilerek protein bantları görülebilir. 80 ve 55 kDa zar kesmek için bir kılavuz olarak bantlar ve protein merdiven kullanın. Not: Adım 4.4 örnekleri 16 x 18 cm 34 kDa aşağı 2 jelleri çalıştırılabilir, belirtilen pozisyonlarda membran kesip kesip değil kolay şekildeilgi bantları. Alternatif olarak, çeşitli jeller zarı kesme önlemek için aynı örnekleri ile çalıştırılabilir. </li><li> % 0.05 Tween 20 (TBST, Tablo 1) ihtiva eden Tris-tamponlu tuzlu su ile Ponceau S boyama yıkayın. Tamamen zarları kapsayan% 5 yağsız süt ile TBST ilave edin. 1 saat boyunca çalkalama ile, oda sıcaklığında membranlar inkübe edin. </li><li> membranlar inkübe O / N ADAR1 (110 ve 150 kDa), fosfo-PKR (62 kDa) ve fosfo-IRF3 (47-kDa) karşı 1,000&#39;de 1 seyreltilmiş% 3 sığır serum albümini ile TBST (BSA) ve antikorlarda, sırasıyla membran, üst orta ve alt parçalar için. </li><li> Membranlar TBST ile 5 dakika için 5 kat ve 1 saat (5,000 içinde 1 seyreltilmiş),% 5 yağsız süt ve peroksidaz-etiketli keçi anti-tavşan sekonder antikor ile TBST içinde inkübe yıkayın. </li><li> TBST ile 5 dakika boyunca membranlar 5x yıkayın ve üreticinin talimatlarına göre filmler bantları görselleştirmek için elektrokemiluminesan (ECL) çözümü uygulayın. </li><li> filmler protein bantları görselleştirmek sonra bir antikor sıyırma çözeltisi ile 10 dakika boyunca zarın, orta ve alt parçaları yıkayın. ve IRF3 (500 &#39;de 1 olarak), toplam PKR karşı% 3 BSA ile TBST içinde membranlar O / N ve antikor inkübe (1.000 de 1), sırasıyla, orta ve alt parçalar için. Yineleyin 4.8 adımları. ve 4.9. PKR membran (orta), anti-tavşan ikincil antikoru yerine peroksidaz etiketli keçi anti-fare kullanılmıştır. </li><li> TBST ile 5 dakika için membran 5x alt parça yıkayın (5,000 içinde 1 seyreltilmiş) aktine karşı bir antikor ile 1 saat boyunca% 3 BSA ile TBST içinde membran inkübe edin. Yineleyin 4.8 adımları. ve 4.9. anti-tavşan sekonder antikor yerine peroksidaz etiketli keçi anti-fare kullanılmıştır. Farklı test RNA ve pozitif kontrol karşılaştırma sonuçlarının bir örneği Şekil 5&#39;te sağlanmaktadır. Kullanılacak özel antikorlar için Malzemelerin tabloya bakınız. </li></ol>

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F., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+translation+and+its+regulation+by+cellular+factors+PKR+and+PACT.">HIV-1 translation and its regulation by cellular factors PKR and PACT.</a> Virus Res. 193, 65-77 (2014).</li><li>ter Brake, O., Konstantinova, P., Ceylan, M., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+HIV-1+with+RNA+interference:+a+multiple+shRNA+approach.">Silencing of HIV-1 with RNA interference: a multiple shRNA approach.</a> Mol Ther. 14, 883-892 (2006).</li><li>Naito, Y., 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=Optimal+design+and+validation+of+antiviral+siRNA+for+targeting+HIV-1.">Optimal design and validation of antiviral siRNA for targeting HIV-1.</a> Retrovirology. 4, 80 (2007).</li><li>Mandal, D., Feng, Z., Stoltzfus, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+RNA+splicing+and+inhibition+of+HIV-1+replication+induced+by+modified+U1+small+nuclear+RNAs.">Excessive RNA splicing and inhibition of HIV-1 replication induced by modified U1 small nuclear RNAs.</a> J Virol. 84, 12790-12800 (2010).</li><li>Chang, 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=Enhanced+expression+and+HIV-1+inhibition+of+chimeric+tRNA(Lys3)-ribozymes+under+dual+U6+snRNA+and+tRNA+promoters.">Enhanced expression and HIV-1 inhibition of chimeric tRNA(Lys3)-ribozymes under dual U6 snRNA and tRNA promoters.</a> Mol Ther. 6, 481-489 (2002).</li><li>Chang, L. J., Liu, X., He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+siRNAs+targeting+multiple+highly+conserved+RNA+sequences+of+human+immunodeficiency+virus+type+1.">Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1.</a> Gene Ther. 12, 1133-1144 (2005).</li><li>Daniels, S. M., 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=HIV-1+RRE+RNA+acts+as+an+RNA+silencing+suppressor+by+competing+with+TRBP-bound+siRNAs.">HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs.</a> RNA Biol. 12, 123-135 (2015).</li><li>Castanotto, 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=Combinatorial+delivery+of+small+interfering+RNAs+reduces+RNAi+efficacy+by+selective+incorporation+into+RISC.">Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC.</a> Nucleic Acids Res. 35, 5154-5164 (2007).</li><li>Vickers, T. A., Sabripour, M., Crooke, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+adaptors+result+in+reduction+of+multiple+pre-mRNA+species+principally+by+sequestering+U1snRNP.">U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP.</a> Nucleic Acids Res. 39, e71 (2011).</li><li>Tai, C. J., Li, C. L., Tai, C. J., Wang, C. K., Lin, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Viral+Entry+Assays+for+the+Identification+and+Evaluation+of+Antiviral+Compounds.">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J Vis Exp. , (2015).</li><li>Riss, T. L., et al., Sittampalam, G. 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> Assay Guidance Manual. , (2004).</li><li>Reynolds, 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=Induction+of+the+interferon+response+by+siRNA+is+cell+type-+and+duplex+length-dependent.">Induction of the interferon response by siRNA is cell type- and duplex length-dependent.</a> Rna. 12, 988-993 (2006).</li><li>Whitehead, K. A., Dahlman, J. E., Langer, R. S., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+or+stimulation?+siRNA+delivery+and+the+immune+system.">Silencing or stimulation? siRNA delivery and the immune system.</a> Annu Rev Chem Biomol Eng. 2, 77-96 (2011).</li></ol>

The authors have nothing to disclose.

Tanımlanan HIV-1 üretimi deneyi HEK293T hücreleri (Şekil 2) kullanılarak gerçekleştirilmiştir ve etkili bir ribozim 13 shRNA 10,29, siRNA 30 ve U1i RNA 11,31 hedef sitesi HIV-1 RNA taramak için kullanılan deneyler benzer edildi. HIV-1 üretimini ölçmek için çeşitli yöntemler kullanılarak, çalışmaların çoğu, aday RNA&#39;lar ile bir HIV-1 ifade plazmidinin Müşterek transfeksiyon sonrasında, virüs üretimini 48 saat ölçtük. HIV-1 ü...

Mevcut HIV-1 terapileri sınırlaması, kronik hastalığın ilerlemesini engellemek için de uygulanabilir olmasıdır. HIV-1 karşı T lenfosit veya hematopoietik kök hücre nakli, ilaç tedavisi 1,2 yokluğunda HIV-1 replikasyonu, uzun süreli kontrolünü sağlama potansiyeline sahiptir ve aynı zamanda, bir HIV-1 ilacı elde etmek için etkili bir yaklaşım olabilir 3.. HIV-1 replikasyonu dirençli hücre oluşturmak için bir yolu, otolog transplant 4 sırasında enfekte bireyin hücrelerine anti-HIV-1 RNA veya peptitler kodlayan bir ya da daha fazla gen eklemektir. Çeşitli aday anti-HIV-1 genlerinin herhangi bir tek genin HIV-1 direnç gelişmesini önlemek için, iki adet 5 ya da üç 6 kombinasyonları bazı giren klinik çalışmalarda dizayn edilmiştir. Anti-HIV-1 RNA immün yanıtları ortaya çıkarmak için düşük potansiyel ve onlar için kombine gen terapisi için en iyi adaylar arasındaÇok kısa bir gen sekanslarından transkribe edilmektedir. Bazı anti-HIV-1 RNA, viral giriş ve bütünleşmesini hedeflemek üzere dizayn edilmiştir. Bununla birlikte, en anti-HIV-1 RNA, viral yaşam çevriminde sonrası entegrasyon adımlar (Şekil 1) hedefler. Sonrası entegrasyon inhibitörleri ribozimler 7, 8 ve shRNAs U1i RNA&#39;lar 9 olarak, HİV-1 düzenleyici proteinleri tat veya rev 1 ve antisens dayalı RNA hedef HIV-1 RNA farklı sitesi hedef, yem RNA içerir. Anti-HIV-1 RNA etkinliğini karşılaştırmak için kullanılmış olan yöntemler genlerin aday RNA&#39;lar kodlayan ve viral geçici aday RNA ifade eden plasmidler ile transfekte edilen hücrelerde üretim ve HIV-1 ifade plazmid ölçüm ile dönüştürülmüş hücrelerin viral replikasyonu izlenmesini, 10 -13. Daha önce yeni bir ribozim, hedef siteye 13-15 HIV-1 RNA taranması için bir HIV-1 üretimi deneyi kullandık. Bu yöntemler beri bir RNA biçimini optimize etmek için rafine edilmişgirişim molekülü, bir shRNA plazmid DNA&#39;sından ifade edilen ya da sentetik bir siRNA 16 olarak verilir. Deney insan embriyonik böbrek (HEK) 293T hücrelerinde olgun virüs üretimini ölçer ve (Şekil 1), HIV-1 replikasyon döngüsünde sonrası entegrasyon evreleri hedefleyen önleyicilerinin etkilerini karşılaştırmak için kullanılabilir. Ön entegrasyon basamakları hedefleyen inhibitörleri, böyle bir tzm-bl hücre enfektivite tahlil 17 gibi alternatif deneyler antiviral etkinliğini değerlendirmek için gereklidir. Klinikte anti-HIV-1 RNA teslimi için önemli güvenlik kaygıları, insan RNA veya protein ve doğuştan gelen bağışıklık sensör aktivasyonu üzerindeki potansiyel hedef dışı etkiler bulunmaktadır. Anti-HIV-1 siRNA&#39;lar toksisitesini değerlendirmek için, farklı hücre hatları 16 bir hücre canlılığı deneyi kullandık. Ayrıca, iki iplikçikli bir RNA bağışıklık sensörler aktivasyonu ölçülmüştür RNA aktive edilmiş protein kinaz R (PKR) reseptör 3 (TLR3) Toll benzeri gibi exinterferon pression geni ADAR1 P150 uyarılmış. Bu deneyler, anti-HIV-1 RNA etkinliği hücre canlılığı veya immün sensör aktivasyon dolaylı etkileri nedeniyle olmadığını teyit etmek için kullanılabilir. Ayrıca, daha da geliştirilmesi, potansiyel toksisite aday RNA&#39;ların hariç yararlıdır. Aşağıdaki protokollerde, prosedürler yeni tedavi RNA&#39;lar belirlemek ve mevcut biçimi açıklanmıştır optimize etmek. Yöntemleri, HIV-1 replikasyonu tarama RNA temelli sonrası entegrasyon inhibitörleri için faydalı olan ve bu tür viral RNA 18 ya da CRISPR Rev aracılı ihracat hedefleme küçük moleküller gibi diğer sonrası entegrasyon inhibitörlerinin taranması için adapte edilebilir / tasarlanan Cas sistemleri entegre hedef HIV-1 DNA&#39;sı 19.

<table><tbody><tr><td>DMEM HyClone</td><td>GE Healthcare</td><td>SH30243.01</td><td></td></tr><tr><td>FBS HyClone</td><td>GE Healthcare</td><td>SH30396.03</td><td></td></tr><tr><td>Penicillin/Streptomycin Gibco</td><td>Thermo Fisher</td><td>15140-122</td><td></td></tr><tr><td>Cell culture plates, 96 well, 24 well, 6 well.</td><td>Corning</td><td>353075, 353047, 353043</td><td></td></tr><tr><td>Micro tubes Axygen</td><td>Corning</td><td>311-08-051</td><td></td></tr><tr><td>Low molecular weight Poly I:C</td><td>InvivoGen</td><td>3182-29-6</td><td></td></tr><tr><td>DharmaFECT-1</td><td>Dharmacon</td><td>T-2001-01</td><td>transfection reagent for synthetic RNAs</td></tr><tr><td>TransIT-LT1</td><td>Mirus</td><td>MIR 2300</td><td>transfection reagent for RNA expression plasmids</td></tr><tr><td>Nonidet P40 (NP-40)</td><td>USB</td><td>19628</td><td></td></tr><tr><td>[&lt;sup&gt;32&lt;/sup&gt;P]dTTP</td><td>Perkin Elmer</td><td>BLU505H</td><td></td></tr><tr><td>poly(A) RNA template&amp;nbsp;</td><td>Sigma-Aldrich</td><td>10108626001</td><td></td></tr><tr><td>oligo(dT)&lt;sub&gt;12-18&lt;/sub&gt; DNA primer</td><td>Thermo Fisher</td><td>18418-012</td><td></td></tr><tr><td>DEAE filtermat paper&amp;nbsp;</td><td>Perkin Elmer</td><td>1450-522</td><td></td></tr><tr><td>Microplate scintillation counter</td><td>Perkin Elmer</td><td>1450-024</td><td></td></tr><tr><td>MTT</td><td>Sigma-Aldrich</td><td>M-2128</td><td></td></tr><tr><td>DPBS HyClone</td><td>GE Healthcare</td><td>SH30028.02</td><td></td></tr><tr><td>Microplate spectrophotometer</td><td>Bio-rad</td><td>1706930</td><td></td></tr><tr><td>Lysis buffer tablets</td><td>Roche</td><td>4693159001, 4906837001</td><td>protease and phosphatase inhibitors</td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5415R</td><td></td></tr><tr><td>Bradford reagent</td><td>Bio-rad</td><td>500-0006</td><td></td></tr><tr><td>Gel running chamber</td><td>Hoefer</td><td>SE600</td><td></td></tr><tr><td>Semi-dry transfer cell</td><td>Bio-rad</td><td>1703940</td><td></td></tr><tr><td>Protein ladder EZ-Run</td><td>Thermo Fisher</td><td>BP3603-500</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Bio-rad</td><td>162-0094</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A9647-1006</td><td></td></tr><tr><td>Antibody stripping solution</td><td>Millipore</td><td>2504</td><td></td></tr><tr><td>ECL - Pierce</td><td>Thermo Fisher&amp;nbsp;</td><td>PI32106</td><td></td></tr><tr><td>ADAR1 antibody</td><td></td><td></td><td>from Dr. B.L. Bass</td></tr><tr><td>phospho-T446-PKR antibody</td><td>Abcam</td><td>ab32036</td><td></td></tr><tr><td>phospho-S396-IRF3 antibody</td><td>Cell Signaling</td><td>4947</td><td></td></tr><tr><td>PKR antibody</td><td></td><td></td><td>from Dr. A. Hovanessian</td></tr><tr><td>IRF3 antibody</td><td>Cell Signaling</td><td>11904</td><td></td></tr><tr><td>Actin antibody</td><td>Millipore</td><td>MAB1501</td><td></td></tr><tr><td>Peroxidase-labeled goat anti-rabbit</td><td>KPL</td><td>474-1506</td><td></td></tr><tr><td>Peroxidase-labeled goat anti-mouse</td><td>KPL</td><td>474-1806</td><td></td></tr><tr><td>Ponceau S&amp;nbsp;</td><td>Sigma-Aldrich</td><td>6226-79-5</td><td></td></tr></tbody></table>

evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Prosedürleri genel şematik bir üç test RNA ve Şekil 2B&#39;de sağlanan bir kontrol RNA için bir örnek transfeksiyon planı, Şekil 2 &#39;de gösterilmiştir. Viral üretimi ve hücre yaşayabilirliği tahlilleri için, her bir test yapı için Okuması bir negatif kontrol normalize edilir. Her test RNA bitişiğindeki negatif kontrol normalize böylece çoğaltır, setler halinde transfekte edilir. Bu durum, örneğin, negatif kontroller tüm bi...

Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Bir gen ya da ilaç terapisinde kullanım için, HIV-1 üretim yapan RNA&#39;lar etkinlik ve toksisite Değerlendirilmesi

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

evaluation-efficacy-toxicity-rnas-targeting-hiv-1-production-for-use

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.8K Görüntüleme.  Lady Davis Institute for Medical Research. Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones. 

Video: Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

Tedavi Oncolytic virusu modüle ana faktörleri tanımlamak için genom çapında RNAi tarama

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Kanser Araştırmaları

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

Hücre tipi siRNA teslimat için özel anti-HIV gp120 aptamers Geliştirme

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

İmmünoloji ve Enfeksiyon

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

Kanser gen Fonksiyon Eğitim için DNA vektör tabanlı RNA Girişim

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

Tıp

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA Virüslerinin geniş spektrumlu Kimyasal Önleyicileri için yüksek verimli tarama

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV ters transkriptaz ve integraz inhibitörleri hızlı taranması

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

RNAi Efektör Bakteriyel Teslim Tarihi: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Biyoloji

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

Picornavirüs tropizm MicroRNA tabanlı Yönetmelik

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

Vitro değerlendirme HIV transkripsiyon ve yapıştırma ajanları geri gecikme süresi yüksek işlem hacmi

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Floresans tarafından viral RNA tespit edilmesi<em&gt; In situ</em&gt; Hibridizasyon (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. Although combinational antiretroviral therapy (cART) has been successful in suppressing viral replication, it cannot completely eradicate the reservoir from HIV-infected individuals. A safe and effective cure strategy for HIV infection will require multipronged methods, and therefore the advancements of animal models for HIV-1 infection are pivotal for the development of HIV cure research. Humanized mice recapitulate key features of HIV-1 infection. The humanized mouse model can be infected by HIV-1 and viral replication can be controlled with cART regimens. Moreover, cART interruption results in a prompt viral rebound in humanized mice. However, administration of cART to the animal can be ineffective, difficult, or toxic, and many clinically relevant cART regimens are unable to be optimally utilized. Along with being potentially unsafe for researchers, administration of cART by a commonly used intensive daily injection procedure induces stress by physical restraint of the animal. The novel oral cART method to treat HIV-1 infected humanized mice described in this article resulted in suppression of viremia below the detection level, increased rate of CD4+ restoration, and improved overall health in HIV-1 infected humanized mice.

This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

HIV-1 Enfekte İnsanlaştırılmış Farelerde Oral Kombinasyonel Antiretroviral Tedavi

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. ...

Bir gen ya da ilaç terapisinde kullanım için, HIV-1 üretim yapan RNA'lar etkinlik ve toksisite Değerlendirilmesi

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