Yazarlar agroinfiltration geliştirilmesi ve in vitro montaj deneylerinde değerli önerileri için laboratuvar birkaç üyelerine teşekkür etmek istiyorum. Bu çalışma, California Üniversitesi&#39;nden bir hibe ile finanse edildi. Bu çalışma, Ulusal Bilim Vakfı (CBET-1.144.237) tarafından verilen bir hibe bölgelerinde desteklenmiştir.

1. Bitki materyali <ol><li> Encapsidation deneyinde kullanılacak Nicotiana benthamiana bitkilerin yapraklı 4 aşama (yaklaşık 3-4 hafta yaşında bitkiler) olmalıdır. </li></ol> 2. Fonksiyonel viral genomun bileşenlerinin Teslimat ve ifade agroinfiltration tarafından hücreleri bitki <ol><li> 1. Gün: Agrobacterium suşu (örn. EH 105 veya GV 3101) pCass-BMV RNA 1, BMV RNA 2 ve RNA 3 BMV 1,2 barındıran 50 kanamisin mg / ml ile desteklenmiş LB agar yetiştirilmektedir (için seçer pCASS vektör) ve rifampisin 100mg/ml () Agrobacterium için seçer. Kuluçka, iki gün süre ile 28 ° C sıcaklıkta yapılmaktadır. </li><li> 3. Gün: 50 mg / kanamisin ve 28 az 1 gün boyunca 100 mg / rifampisin ml ° 250 RPM hızında ayarlanmış orbital karıştırıcıda C ml&#39;lik LB suyu 2 ml&#39;lik LB agar plakadan İnokülasyon tek koloni. </li><li> 4. Gün: LB suyu takviyesi 50 ml inoküleed 50 ile mg / kanamisin ve rifampicin ve 100 mg / ml, 10 mM MES pH 5.6 ml ve 100 mM 28 azından 16 saat boyunca Erlenmeyer şişesinde bir 250 ml&#39;lik kültür biri ml acetosyringone çalkalamalı ° C&#39;de 250 RPM&#39;de . </li><li> 5. Gün: 4 ° C&#39;de 5000 rpm&#39;de 10 dakika boyunca bir meşe sırtı tüp veya steril Falcon tüpü ve santrifüj (OD 600 1.0 ulaştığında) kültür aktarın </li><li> Bu aşama, bir antibiyotik tamamen çıkarılması sağlamak üzere bir kez daha tekrarlama, 4 ° C&#39;de 5.000 rpm&#39;de 10 dakika boyunca 10 mM MgCl2 ve santrifüj 10 ml pelet içinde çözülür. </li><li> 10 mM MgCl2, 10 mM MES (pH 5.6) ihtiva eden 10 ml tampon içinde askıya pelet. </li><li> BMV RNA 1, RNA 2 ve RNA 3 her kültür için OD 600 ölçün ve 10 mM MgCl2 ve 10mM MES (pH 5.6) kullanılarak 0.1 OD 600 ayarlayın. </li><li> 1 ml her üç 0.1 OD 600 kültür süspansiyonlar karıştırın. </li><li> Acetosyringone 100 mM, mix gent eklely ve 3 saat boyunca oda sıcaklığında karışıma bozulmamış tutmak. </li><li> 1 mL kültür süspansiyon çizin. İğne olmadan şırınga. </li><li> N. 2-3 iyi genişletilmiş yaprak eksen dışı tarafına yukarıda kültür süspansiyon Infiltrate yavaşça yaprak eksen dışı yüzeyinin yarısına şırınga basarak benthamiana (evre 5 yaprak, 3-4 haftalık bir bitki). </li><li> Diğer yaprakları için bu sızma işlemi tekrarlayın. </li><li> 24 ° C ile yeşil evde infiltre bitkiler tutun </li><li> Hasat 4 gün sonrası infiltrasyon (dpi) yaprakları 3 sızmış. </li></ol> 3. BMV virionlar saflaştırılması <ol><li> N. toplayın benthamiana üç vahşi tip BMV agrotransformants ihtiva eden bir karışım ile agroinfiltrated bırakır. </li><li> 0,08 M ​​MgAc, pH 4.5 ve kullanılmadan önce ilave edilir β-mercaptoethanol ile 1/100 hacim; BMV ekstraksiyon tamponu (0.5 M NaAc arasında eşit hacimde (w / v) içeren bir steril havaneli ve havanda yapraklar öğütmek) 3. Virüs verimi en üst düzeye çıkarmak için, taşlama kolay ve verimli hücre parçalama kolaylaştırır asit yıkanmış kum (~ 0.5-1 gr) eklenmesi önerilir. </li><li> Muslin kumaş 2-3 katmanları üzerinden yaprak süzülür ve telaş toplamak. </li><li> Yine yaprak malzemenin ilk ağırlığına BMV ekstraksiyon tamponu eşit hacmi ile muslin kumaş üzerine tutulan kısmı eziyet. </li><li> Muslin kumaş kullanılarak filtrasyon işlemi tekrarlayın. Bu adımlar, 4 ° C de yapılmalıdır </li><li> Bir santrifüj tüpüne aktarılır ve süzüntü çözeltinin süspansiyon renk açık yeşil olana dek oda sıcaklığında 5 dakika boyunca ön-soğutulmuş ve kloroform vorteks ile eşit hacimde (veya titreme) ilave edilir. </li><li> 4 at 12,000 RPM 15 dakika emülsifiye çözüm santrifüjleyin ° C </li><li> Sulu faz, toplamak ve kalan kloroform kaldırmak için manyetik bir karıştırma düzeni üzerinde 30 dakika boyunca karıştırılır. </li><li> Steril bir ultrasantrifüjdeki tüpüne süspansiyon aktarın. </li><li&gt; 1 ml Underlay. Bir yastık 3 olarak% 10 sukroz (virüsü ekstraksiyon tampon içinde hazırlanmıştır). </li><li> 4 de, bir Beckman ultrasantrifüjdeki 3 saat süreyle 30,000 rpm&#39;de santrifüje ° C. </li><li> Süpernatant atın ve (steril saf su ile 1/10 süspansiyon tampon seyreltik BMV ekstraksiyon tamponu hazırlamak için) BMV süspansiyon tamponu istenilen hacimde pelet askıya. </li><li> Yukarıda adım ikinci kısmen saflaştırılmış virion hazırlanması 4 ° C&#39;de 28.000 rpm&#39;de 150 dakika için% 10-40 sukroz yoğunluk gradyanı santrifüje tabi tutulur </li><li> El ile veya bir parçalama tarafından virüs bant toplayın. Virüs bantlı beyaz ışık aydınlatması altında mavi görünecektir. </li><li> Virüs süspansiyon tamponu ile en azından% 50 virüsü Örnek içeren sukroz çözeltinin. </li><li> 4 bir Beckman ultrasantrifüjdeki 30,000 rpm&#39;de 3 saat boyunca seyreltilmiş virüs içeren sakaroz çözeltisini santrifüjleyin ° C </li><li> Son olarak Vir istenen bir hacmi yüksek oranda saflaştırılmış virüs pelet askıyabize süspansiyon tampon. </li><li> OD 260 spektrofotometre kullanılarak virion (mg / ml) konsantrasyonu ölçülür. </li></ol><img alt="Denklem 1" src="/files/ftp_upload/3645/3645eq1.jpg" /> Not: RNA içeriğine dayanılarak, BMV için yok olma katsayısı 5 4 olur. <ol start="19"><li> TEM ile görüntüleyerek virionlar saflığını (Şekil A) doğrulayın </li></ol> 4. In vitro montaj için kapsid protein alt biriminin hazırlanması <ol><li> 5 1x virüsü ayrışma tamponu (; 50 mM Tris HCI, pH 7.5;; 1.0 mM EDTA 1.0 mM DTT ve 0.5 mM PMSF 0.5 M CaCI2), 1 litre hazırlayın. </li><li> Sambrook et al 6&#39;ya göre diyaliz membran hazırlayın. </li><li> Bir diyaliz torbasına virüs saflaştırılmış gerekli konsantrasyonu dağılır. </li><li> 1x virüs ayrışma tampon içeren behere virüs içeren diyaliz çantası yerleştirin. </li><li> 4 ° C de 24 saat whil Dialyzee karıştırma. </li><li> 4 azından 15 dakika süreyle 12,000 x g&#39;de 24 saat ve sonra santrifüj diyaliz torbasından çözeltisi Collect ° C. Bu ayrışmış virionlar gelen BMV RNA ayıracaktır. </li><li> 4 pelet ° C herhangi bir un-ayrışmış virüs parçacık 3 saat Beckman santrifüj 35,000 rpm süpernatan ve santrifüj toplayın. </li><li> Süpernatant toplayın. </li><li> Bu aşamada ayrışmış kapsid protein alt biriminin herhangi bir tehlike virion RNA alınması zorunludur. Bunu yapmak için, adım 8 yüzen bir yüksek hızlı santrifüj (3 saat az 30.000 rpm ardından herhangi bir RNA (RNA 1x montaj tampon, aşağıya bakınız) eklemeden kapsid protein alt biriminin reassembly gece boyunca bir tur tabi tutulmalıdır 4 ° C) pelet bir araya virionlar için. Bu reassembly adımdan sonra, süpernatant içeren kat protein alt biriminin herhangi bir kalıntı kirletici RNA ücretsiz olacaktır. Bununla birlikte, bu sağlamak için, bu v ek bir gerçekleştirmek için tavsiye edilirreassembly RNA 1x montaj tampon kullanarak tek kat protein alt birimleri ile itro. Reassembly gece boyunca sonra, hazırlık (TEM kullanarak doğrulamak) monte virionlar arındırılmış olmalıdır. </li><li> OD 254 ve 280 nm&#39;de ölçerek veya Bradford deneyi gibi diğer yöntemler ile kapsid proteini alt birimlerinin konsantrasyonunu belirlemek. </li><li> % 12-15 disosiye kapsid protein alt biriminin bütünlüğünü belirlemek için western blot takip SDS-PAGE gerçekleştirin. </li></ol> 5. RNA in vitro montajında ​​virionlar içeren <ol><li> Virionlar içine tekrar monte edilmesi RNA transkript hazırlayın ve konsantrasyonu 7 hesaplayabilirsiniz. </li><li> Kapsid protein alt biriminin karıştırın ve 1:5 (ağırlık / ağırlık) 5 oranında transkript RNA. </li><li> Bir diyaliz torbasına yukarıdaki karışımı dağıtın ve doğru bir sızıntıları önlemek için güvenli. </li><li> 1.000 ml hazırlayın. 5 RNA 1x montaj tamponu (; 50 mM Tris-HCl, pH 7.2;; 10 mM KCl 5 mM MgCl2, 1.0 mM DTT, 50 mM NaCl). </li><li> Behere 1x montaj tampon karşı reaksiyon karışımını içeren diyaliz torbası koyun ve karıştırın. </li><li> Nazik karıştırılarak 24 saat boyunca 4 ° C&#39;de yeniden birleştirme reaksiyonu Dialyze. </li><li> 24 saat sonra diyaliz torbasından karışımı toplayın ve RNA montaj tamponu 1.5 ml ekleyin. </li><li> 30 dakika daha düşük hızda (2000 x g) santrifüj edilerek bir Centricon-100 sütunu üzerinden bu karışım geçmektedir. </li><li> 30 dakika süre ile 4 ° C&#39;de montaj tamponu ile bir kez 1.5 ml kolon yıkanır. </li><li> Iki kez yukarıdaki yıkama adımı tekrarlayın. </li><li> Son olarak, 4 azından 10.000 g&#39;de santrifüje edilerek yeniden monte virionlar elüt edilmesi ° C&#39;de 5 dakika için. </li><li> OD 260 nm virion konsantrasyonu tahmin. </li></ol> 6. Optik Viral Ghosts of Fabrikasyon (OVGs) <ol><li> Bir istenilen konsantrasyon oranı (bu raporda kullanılan yeşil ve kapsid protein İndosiyanin ağırlık oranı 1:10 idi) az saflaştırılmış kapsid protein green (ICG) 8 İndosiyanin ekle<html> d pipetleme ile iyice karıştırın. </li><li> 24 saat boyunca 4 ° C&#39;de OVG montaj tamponu (ve 1 mM DTT, pH4.8, 50 mM NaAc;; 1 mM EDTA 1 M NaCl) karşı kapsid protein ve ICG solüsyonu Dialyze. </li><li> 24 saat sonra, 4 &#39;de 1 saat için 90,000 rpm&#39;de diyaliz torbası (12 kDa gözenek boyutu) ve santrifüj gelen çözelti toplamak ° C (Şema B) </li><li> 4 ° C&#39;de vorteks veya bir gecede BMV süspansiyon tampon kendisi tarafından askıya izin vererek Süpernatantı ve BMV süspansiyon tampon pelet askıya </li><li> TEM (Şekil C) OVGs morfolojisini doğrulayın. </li><li> 240 nm spektrofotometre (Cary 50, Varian Inc) kullanarak 950 nm absorbansı ölçülür; ICG varlığı 700 nm ve 790 nm 8 civarında imza absorbans pikleri ile teyit edilir. (Şekil D) </li><li> ~ 700 nm ve 800 nm &#39;~ tipik ICG floresans zirveleri spectrofluorometer kullanılarak OVG çözeltisinin 620 nm eksitasyon (Şekil D) (Fluorolog 3, Jobin-Yvon) üzerine görülebilir. </li></ol>_title &quot;&gt; 7. Temsilcisi Sonuçlar <img alt="Şekil A" src="/files/ftp_upload/3645/3645figA.jpg" /> N. arınmış olumsuz lekeli BMV virionlar Şekil A. TEM görüntüsü benthamiana bitkiler üç vahşi tip BMV agroconstructs (ölçekli bar = 100 nm) oluşan bir karışım ile agroinfiltrated. <img alt="Şekil B" src="/files/ftp_upload/3645/3645figB.jpg" /> In vitro Şekil B. Pelet yüksek hızda santrfüj uygulanmıştır ihtiva OVGs ICG toplandı. <img alt="Şekil C" src="/files/ftp_upload/3645/3645figC.jpg" /> Olumsuz lekeli ICG içeren OVGs Şekil C. TEM görüntüsü (scale bar = 100 nm). <img alt="Şekil D" src="/files/ftp_upload/3645/3645figD.jpg" /> OVGs Şekil D. Absorbans (Üst) ve floresan (alt) spektrumları. OVG emisyon w için kullanılan dalgaboyu620 nm olarak.

<ol>
	<li>Annamalai, P., Rao, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-independent+expression+of+genome+components+and+capsid+protein+of+brome+mosaic+virus+in+planta:+a+functional+role+for+viral+replicase+in+RNA+packaging.">Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: a functional role for viral replicase in RNA packaging.</a> Virology. 338, 96-111 (2005).</li><li>Annamalai, P., Rofail, F., Demason, D. A., Rao, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-coupled+packaging+mechanism+in+positive-strand+RNA+viruses:+synchronized+coexpression+of+functional+multigenome+RNA+components+of+an+animal+and+a+plant+virus+in+Nicotiana+benthamiana+cells+by+agroinfiltration.">Replication-coupled packaging mechanism in positive-strand RNA viruses: synchronized coexpression of functional multigenome RNA components of an animal and a plant virus in Nicotiana benthamiana cells by agroinfiltration.</a> J. Virol. 82, 1484-1495 (2008).</li><li>Annamalai, P., Rao, A. L., Foster, N., Johansen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Virology Protocols. 451, 251-264 (2008).</li><li>Lane, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bromoviruses.">The bromoviruses.</a> Adv. Virus Res. 19, 151-220 (1974).</li><li>Choi, Y. G., Rao, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+studies+on+bromovirus+capsid+protein.+VII.+Selective+packaging+on+BMV+RNA4+by+specific+N-terminal+arginine+residuals.">Molecular studies on bromovirus capsid protein. VII. Selective packaging on BMV RNA4 by specific N-terminal arginine residuals.</a> Virology. 275, 207-217 (2000).</li><li>Sambrrok, J., Russel, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning. A Laboratory Manual. , (2001).</li><li>Dreher, T. W., Rao, A. L., Hall, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+in+vivo+of+mutant+brome+mosaic+virus+RNAs+defective+in+aminoacylation.">Replication in vivo of mutant brome mosaic virus RNAs defective in aminoacylation.</a> J. Mol. Biol. 206, 425-438 (1989).</li><li>Jung, B., Rao, A. L., Anvari, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+nano-constructs+composed+of+genome-depleted+brome+mosaic+virus+doped+with+a+near+infrared+chromophore+for+potential+biomedical+applications.">Optical nano-constructs composed of genome-depleted brome mosaic virus doped with a near infrared chromophore for potential biomedical applications.</a> A.C.S. Nano. 5, 1243-1252 (2011).</li><li>Voinnet, O., Lederer, C., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+viral+movement+protein+prevents+spread+of+the+gene+silencing+signal+in+Nicotiana+benthamiana.">A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana.</a> Cell. 103, 157-167 (2000).</li><li>Wroblewski, T., Tomczak, A., Michelmore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Agrobacterium-mediated+transient+assays+of+gene+expression+in+lettuce,+tomato+and+Arabidopsis.">Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis.</a> Plant Biotechnol. J. 3, 259-273 (2005).</li></ol>

Biz ifşa hiçbir şey yok.

Burada sunulan Agroinfiltration yaklaşımı yaygın olarak bitki virüsleri, geniş bir aralık uygulanabilir olabilir. Bu yaklaşımın bir işaretidir özelliği aynı hücre-sık bitki virüsleri rutin olarak kullanılan mekanik inokulasyon ile ilişkili büyük dezavantajı birden çok agroconstruct teslim eşitlenir. In vivo ve bir model olarak brom mozaik virüsü kullanılarak in vitro montaj çalışmaları ile verimli bir şekilde yapılabilir Aşağıdaki birkaç ipucu. (i) N.</...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> MES Sodyum tuzları </td><td> Sigma-Aldrich </td><td> M2993 </td></tr><tr><td> Yeşil İndosiyanin </td><td> Sigma-Aldrich </td><td> I2633 </td></tr><tr><td> Beckman ultrasantrifüjdeki </td><td> Beckman Coulter </td><td> Model: L8-70m </td></tr><tr><td> Centricon-100 sütunu </td><td> Millipore </td><td> YM-100 </td></tr><tr><td> Spektrofotometre </td><td> Cary 50, Varian Inc </td><td> Parça numarası 10068900 </td></tr><tr><td> Spectrofluorometer </td><td> Fluorolog 3, Jobin-Yvon. </td><td> Parça numarası FL3-21 </td></tr></tbody></table>

simple and robust in vivo and in vitro approach for studying virus assembly

Insan, hayvan ve bitkiler, yavrulama encapsidation olgun ve kararlı virionlar içine kadar pozitif polariteli RNA genomu patojen olan virüsler belirli bir konak enfeksiyon kurulması sırasında bir kardinal aşamasıdır. Sonuç olarak, encapsidation çalışma know-how bulaşıcı virionlar oluşturmak için virüs montaj düzenleyen mekanizmasının ilişkin bilgileri tercümesini. Bu bilgiler, virüs yaymak ve hastalık kontrolü düşürücü yeni yöntemler oluşturulmasında hayati önem taşımaktadır. Virüs encapsidation in vivo ve in vitro olarak ele alınabilir. In vivo Genom encapsidation makromoleküler etkileşimleri ve hücre içi kompartımanlara içeren oldukça düzenli bir seçici bir süreçtir. Bu nedenle, in vivo virüs encapsidation kapsayan olaylar virüsler çoğalır ve montajı nasıl anlamak için temel bilgi sağlayacaktır incelemek önde gelen eğitim. Son zamanlarda in vitro encapsidation biyomedikal ima alanında araştırma için istismar edilmiştirging ve terapötik uygulamalar. Zarfsız bitki virüslerinin negatif yüklü yabancı madde in vitro encapsidation içinde bir girişim çok daha önde duruyor. Brom mozaik virüsü (BMV), bitkiler için bir zarfsız çok bileşenli bir RNA virüsünün patojenik, in vivo ve in vitro paketleme genomunun çalışmak için bir model olarak sistemi kullanılmıştır. Nicotiana benthamiana bitkilerde encapsidation testlerde, Agrobacterium geçici ifadesi, agroinfiltration dediğimiz aynı hücreye birden çok bileşenden senkronize teslimat ve ifade için verimli ve güçlü bir tekniktir. Bu yaklaşımda, desiredviral mRNA&#39;ların cDNAlarının taşıyan ikili plazmid vektörler taşıyan Agrobacterium tumefaciens bir hücre süspansiyonu 1 ml tek kullanımlık şırınga (iğne olmadan) daha karmaşık bir şey kullanarak hücrelerarası boşluk withina kanadı içine sızmış edilir. Bitki hücrelerine DNA ekleme devri içinde Bu işlem sonucu; T-DNAinsert çekirdeğinde geçici olarak kalır ve daha sonra ev sahibi polimeraz II transkripsiyonu olduğunu, geçici ekspresyonunu. Çıkan mRNA transkript (kep ve polyadenylated) sonra çeviri için sitoplazma ihraç edilmektedir. Inkübasyon yaklaşık 24 ila 48 saat sonra, infiltre yaprakların bölümleri microscopyor biyokimyasal analizler yapılabilir. Agroinfiltration eşzamanlı olarak transfekte edilmesi için hücreler, çok sayıda (yüz binlerce) verir. In vitro encapsidation içinde için, BMV arındırılmış virionlar santrifüjle RNA ve un-ayrışmış virionlar uzaklaştırılması ile devam ayrışma tampon içeren kalsiyum klorür karşı diyaliz tarafından kapsid proteini içine ayrışmış bulunmaktadır. Genom tüketilmiş kapsid proteini alt birimden daha sonra arzu edilen viral genom bileşenleri veya bu tür boya indosiyanin gibi viral olmayan bileşenleri ile yeniden birleştirilen edilir.

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Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

Ile bitki hücreleri için birden fazla viral bileşenlerinin teslim senkronize etmek için basit, verimli ve sağlam bir yol<em&gt; Agrobacterium</em&gt;-Aracılı geçici ifade tarif edilmektedir. Bu yaklaşım, çoğaltma çalışmaları için müsait, encapsidation izledi<em&gt; In vitro</em&gt; Reassembly genomuna viral olmayan bileşenleri biyomedikal uygulamaları için uygun optik viral hayaletler tüketilmiş.

Basit ve Sağlam In vivo Ve In vitro</emVirüs Meclisi Öğrenmenin> Yaklaşım

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

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Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

12.1K Görüntüleme.  University of California, Riverside. Ile bitki hücreleri için birden fazla viral bileşenlerinin teslim senkronize etmek için basit, verimli ve sağlam bir yol Agrobacterium-Aracılı geçici ifade tarif edilmektedir. Bu yaklaşım, çoğaltma çalışmaları için müsait, encapsidation izledi In vitro Reassembly genomuna viral olmayan bileşenleri biyomedikal uygulamaları için uygun optik viral hayaletler tüketilmiş.

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Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Alexa Fluor Etiketleme Dang Virüs görselleştirme

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

İmmünoloji ve Enfeksiyon

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

Bir Nöro-İnvazif encephalitic Virüs Vivo Görüntüleme Sistemleri (IVIS) Algılama

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

A Novel<em&gt; In vitro</em&gt; İnsan Tüm Kan ve Endotelyumunun Arasındaki Etkileşimler incelenmesi için Model

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

Batı Nil Virüsü Mutant Soy Enfeksiyon MyD88-aracılı Hücresel Bağışıklık Tepki Vitro Analizi

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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 ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Gradient Santrifüj tarafından İnfluenza A Virüsü Kapsidlerin Vitro Demontaj içinde

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

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 ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Terapötik Antikorlar Arasındaki Hedef Bağlanma ve CDC İndüksiyonunu Karşılaştırmak İçin İn Vitro Yöntemler: Biyoeşdeğerlik Analizindeki Uygulamalar

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

NK ve kronik aktif Epstein - Barr virüsü enfeksiyonu olan hastalarda T hücre satırlarından kurmak için etkili ve basit bir yöntem

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

Basit Akış Sitometresi Vitro antikor bağımlı Enhancement-in dang virüs Zika virüs nekahat Serum kullanarak belirlemek için tahlil dayalı

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Basit ve Sağlam In vivo Ve In vitro Yaklaşım