نود أن نعترف جبهة الخلاص الوطني للتمويل (CBET-0846259).

<ol>
	<li>Banerjee, P. S., Ostapchuk, P., Hearing, P., Carrico, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoselective+Attachment+of+Small+Molecule+Effector+Functionality+to+Human+Adenoviruses+Facilitates+Gene+Delivery+to+Cancer+Cells.">Chemoselective Attachment of Small Molecule Effector Functionality to Human Adenoviruses Facilitates Gene Delivery to Cancer Cells.</a> J. Am. Chem. Soc. 132, 13615-13617 (2010).</li><li>Banerjee, P. S., Carrico, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoselective+Modification+of+Viral+Proteins+Bearing+Metabolically+Introduced+"Clickable"+Amino+Acids+and+Sugars.">Chemoselective Modification of Viral Proteins Bearing Metabolically Introduced "Clickable" Amino Acids and Sugars.</a> Methods Mol. Biol. 751, 55-66 (2011).</li><li>Waehler, R., Russell, S. J., Curiel, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+targeted+viral+vectors+for+gene+therapy.">Engineering targeted viral vectors for gene therapy.</a> Nat. Rev. Genet. 8, 573-587 (2007).</li><li>Magnusson, M. K., Hong, S. S., Henning, P., Boulanger, P., Lindholm, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Retargeting+of+Adenovirus+Vectors:+Functionality+of+Targeting+Ligands+and+Their+Influence+on+Virus+Viability.">Genetic Retargeting of Adenovirus Vectors: Functionality of Targeting Ligands and Their Influence on Virus Viability.</a> J. Gene Med. 4, 356-370 (2002).</li><li>Henning, P., Lundgren, E., Carlsson, M., Frykholm, K., Johannisson, J., Magnusson, M. K., TÃ¥ng, E., Franqueville, L., Hong, S. S., Lindholm, L., Boulanger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+Type+5+Fiber+Knob+Domain+has+a+Critical+Role+in+Fiber+Protein+Synthesis+and+Encapsidation.">Adenovirus Type 5 Fiber Knob Domain has a Critical Role in Fiber Protein Synthesis and Encapsidation.</a> J. Gen. Virol. 87, 3151-3160 (2006).</li><li>Zhang, M. M., Tsou, L. K., Charron, G., Raghavan, A. S., Hang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+fluorescence+imaging+of+dynamic+S-acylation+and+protein+turnover.">Tandem fluorescence imaging of dynamic S-acylation and protein turnover.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 8627-8632 (2010).</li><li>Banerjee, P. S., Ostapchuk, P., Hearing, P., Carrico, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unnatural+amino+acid+incorporation+onto+adenoviral+(Ad)+coat+proteins+facilitates+chemoselective+modification+and+retargeting+of+Ad+type+5+vectors.">Unnatural amino acid incorporation onto adenoviral (Ad) coat proteins facilitates chemoselective modification and retargeting of Ad type 5 vectors.</a> J. Virol. 85, 7546-7554 (2011).</li><li>Hong, V., Presolski, S. I., Ma, C., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+Optimization+of+Copper-Catalyzed+Azide-Alkyne+Cycloaddition+for+Bioconjugation.">Analysis and Optimization of Copper-Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation.</a> Angew. Chem. Int. Ed. 48, 9879-9883 (2009).</li><li>Best, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+Chemistry+and+Bioorthogonal+Reactions:+Unprecedented+Selectivity+in+the+Labeling+of+Biological+Molecules.">Click Chemistry and Bioorthogonal Reactions: Unprecedented Selectivity in the Labeling of Biological Molecules.</a> Biochemistry. 48, 6571-6584 (2009).</li><li>Agard, N. J., Prescher, J. A., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Strain-Promoted+[3+++2]+Azide-Alkyne+Cycloaddition+for+Covalent+Modification+of+Biomolecules+in+Living+Systems.">A Strain-Promoted [3 + 2] Azide-Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems.</a> J. Am. Chem. Soc. 46, 15046-15047 (2004).</li><li>Dommerholt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Readily+Accessible+Bicyclononynes+for+Bioorthogonal+Labeling+and+Three-Dimensional+Imaging+of+Living+Cells.">Readily Accessible Bicyclononynes for Bioorthogonal Labeling and Three-Dimensional Imaging of Living Cells.</a> Angew. Chem. Int. Ed. 49, 9422-9425 (2010).</li><li>Link, A. J., Vink, M. K. S., Tirrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+the+functionalizable+methionine+surrogate+azidohomoalanine+via+copper-catalyzed+diazotransfer.">Preparation of the functionalizable methionine surrogate azidohomoalanine via copper-catalyzed diazotransfer.</a> Nat. Protoc. 2, 1879-1883 (2007).</li></ol>

الإعلان عن أي تضارب في المصالح.

تطور ردود الفعل فوق chemoselective وbioorthogonal، بما فيها تلك التي من أزيدات، هو منطقة التطور السريع للبحوث، وبعد ذلك هناك عدد متزايد من ردود الفعل هذه للاختيار من بينها لتقديم الطلبات bioconjugation. قيدت نحن في نطاق هذا البروتوكول لتشمل اثنين فقط من الأساليب التي تم اختيارها بسبب فا...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td></tr><tr><td> نوع اتش 5 (Ad5) تحتوي على التحوير GFP </td><td> BCBC </td><td> 391 </td></tr><tr><td> الكلى الجنينية البشرية (كلوة الجنين البشري 293) الخلايا </td><td> ATCC </td><td> CRL-1573 </td></tr><tr><td> Dulbecco ومعدلة النسر متوسطة (DMEM)، ارتفاع نسبة الجلوكوز </td><td> إينفيتروجن </td><td> 11965-092 </td></tr><tr><td> Dulbecco ومعدلة النسر متوسطة، لا الميثيونين، لا سيستين (DMEM و الأرصاد الجوية /-السيستئين)، ارتفاع نسبة الجلوكوز </td><td> إينفيتروجن </td><td> 21013-024 </td></tr><tr><td> الأبقار العجل المصل </td><td> إينفيتروجن </td><td> 16170-078 </td></tr><tr><td> بنسلين - الستربتوميسين 100 × الحل (القلم بكتيريا) </td><td> إينفيتروجن </td><td> 15140-122 </td></tr><tr><td> 0.5٪ التربسين-EDTA (10 ×) </td><td&gt; إينفيتروجن </td><td> 15400-054 </td></tr><tr><td> 100 خلية ثقافة صحن مم، الأنسجة ثقافة البوليسترين المعالجة </td><td> دينار بحريني الصقر </td><td> 353003 </td></tr><tr><td> خلية ثقافة CO 2 حاضنة </td><td></td><td></td></tr><tr><td> عدادة الكريات </td><td></td><td></td></tr><tr><td> مجلس الوزراء للسلامة الأحيائية </td><td></td><td></td></tr><tr><td> معقم مرشح محقنة (0.2 ميكرون، خلات السليلوز) </td><td> VWR </td><td> 28145-477 (NA)، 514-0061 (أوروبا) </td></tr><tr><td> أفانتي JE الطرد المركزي </td><td> بيكمان كولتر </td><td> 369001 </td></tr><tr><td> مخروطي أنابيب (50 مل، معقمة) </td><td> دينار بحريني الصقر </td><td> 352098 </td></tr><tr><td> أنبوب، Thinwall، فائقة الوضوح، 13.2 مل، 14 × 89 ملم </td><td> بيكمان </td><td> 344059 </td></tr><tr><td> منبذة فائقة مجهزة 41 SW SW و 60 Rotor </td><td> بيكمان </td><td></td></tr><tr><td> إيبندورف بيوبور الآمن للقفل أنابيب، 1.5 مل </td><td> إيبندورف </td><td> 0030 121.589 </td></tr><tr><td> منبذة 5418 </td><td> إيبندورف </td><td> 5418 </td></tr><tr><td> Centri سبتمبر تدور الترشيح أعمدة هلام </td><td> فصل برينستون </td><td> CS-901 </td></tr><tr><td> إبرة معقمة، قياس 18 </td><td></td><td></td></tr><tr><td> النيتروجين قفاز كيس (إذا CuAAC امؤكسج التي يتعين القيام بها) </td><td></td><td></td></tr><tr><td> قارورة ديوار </td><td></td><td></td></tr><tr><td> النيتروجين السائل </td><td></td><td></td></tr><tr><td> الكهربائي للخلية </td><td></td><td></td></tr><tr><td> فلوري هلام الماسح الضوئي </td><td></td><td></td></tr><tr><td> استعداد جل تريس، حمض الهيدروكلوريك جل </td><td> الحيوي راد </td><td> 161-1105 </td></tr><tr><td> L-Azidohomoalanine </td><td> AnaSpec </td><td> 63669 </td></tr><tr><td> جينا العلوم البيولوجية </td><td> CLK-AA005 </td><td></td></tr><tr><td> Peracetylated N-azidoacetylgalactosamine (ميلان 4 GalNAz) </td><td> إينفيتروجن </td><td> C33365 </td></tr><tr><td> الحرارية العلمية </td><td> 88905 </td><td></td></tr><tr><td> سيغما الدريخ </td><td> A7480 </td><td></td></tr><tr><td> Bathophenanthroline disulfonic حامض (BDA) ملح الصوديوم </td><td> النائب Biomedicals </td><td> 0215011201 </td></tr><tr><td> ثنائي ميثيل سلفوكسيد (DMSO) </td><td></td><td></td></tr><tr><td> الميثانول </td><td></td><td></td></tr><tr><td> تريس قاعدة (تريس (hydroxymethyl) aminomethane) </td><td></td><td></td></tr><tr><td> فوسفات الصوديوم (نا2 هبو 4) </td><td></td><td></td></tr><tr><td> الفوسفات مخزنة المالحة (PBS) </td><td></td><td></td></tr><tr><td> 1M حمض الهيدروكلوريك </td><td></td><td></td></tr><tr><td> الغليسيرول </td><td></td><td></td></tr><tr><td> ألبومين المصل البقري </td><td></td><td></td></tr><tr><td> L-السيستين </td><td></td><td></td></tr><tr><td> L-ميثيونين </td><td></td><td></td></tr><tr><td> SDS (سلفات الصوديوم دوديسيل) </td><td></td><td></td></tr><tr><td> 2-المركابتويثانول </td><td></td><td></td></tr><tr><td> جليكاين </td><td></td><td></td></tr><tr><td> زرقة البروموفينول </td><td></td><td></td></tr><tr><td> السيزيوم كلوريد (CSCL) </td><td></td><td> &amp; Nباهوجان ساماج؛ </td></tr><tr><td> النحاس (I) بروميد (CuBr) </td><td></td><td></td></tr><tr><td> كلوريد الكالسيوم (CaCl 2) </td><td></td><td></td></tr><tr><td> كلوريد البوتاسيوم (بوكل) </td><td></td><td></td></tr><tr><td> كلوريد المغنيسيوم (MgCl 2) </td><td></td><td></td></tr><tr><td> كلوريد الصوديوم (كلوريد الصوديوم) </td><td></td><td></td></tr><tr><td colspan="3"></td></tr><tr><td> آلكاين مسبار ل* CuAAC </td><td></td><td></td></tr><tr><td> طمرة آلكاين </td><td> إينفيتروجن </td><td> T10183 </td></tr><tr><td colspan="3"></td></tr><tr><td> توتر آلكاين مسبار ل* SPAAC </td><td></td><td></td></tr><tr><td> طمرة ديبو آلكاين </td><td> إينفيتروجن </td><td> C10410 </td></tr></tbody></table> * شركات بارزة من الكواشف الكيمياء نقرة ومجموعات تشمل إينفيتروجن، العلوم البيولوجية جينا، شركاء بري، سيغما الدريخ، بحث غلين، انقر فوق أدوات الكيمياء، وBaseclick. ويمكن الاطلاع على مجموعة متنوعة من الأصباغ وآلكاين يغاندس تستهدف في كتالوجات هؤلاء البائعين &quot;.

chemoselective modification of viral surfaces via bioorthogonal click chemistry

وقد تلقت لتعديل جزيئات الفيروس قدرا كبيرا من الاهتمام لإمكانياتها الهائلة في التأثير على العلاج الجيني، والتطبيقات oncolytic وتطوير لقاح. 1،2،3 النهج الحالية لتعديل أسطح الفيروسية، والتي هي في معظمها على أساس علم الوراثة، وغالبا ما تعاني من التوهين تنبيغ. 4،5 الإنتاج الفيروس، والعدوى والخلوية عن طريق chemoselective الكيمياء نقرة، قمنا بتطوير نهج بديل مباشر التي الالتفاف حول هذه القضايا على حد سواء في حين تبقى مرنة للغاية ويمكن الوصول إليها. 1،2 الهدف من هذا البروتوكول هو للتدليل على فعالية استخدام bioorthogonal كيمياء فوق لتعديل سطح من نوع اتش 5 الجسيمات. ويمكن استخدام هذه عملية من خطوتين سواء على المستوى العلاجي 1 أو تحليلي، 2،6 لأنها تسمح لربط chemoselective من الجزيئات التي تستهدف والأصباغ أو الجزيئات الأخرى ذات الاهتمام على البروتيناتمسبقا مع وصفت به أزيد. المزايا الثلاث الرئيسية لهذه الطريقة هي أن (1) وضع العلامات الأيضية يوضح شيئا يذكر لولا تأثير على اللياقة البدنية الفيروسية، 1،7 (2) يمكن أن تستخدم مجموعة واسعة من يغاندس المستجيب، و (3) فهي سريعة بشكل ملحوظ، وموثوق بها يسهل الوصول إليها. 1،2،7 في أول خطوة من هذا الإجراء، ويتم إنتاج جزيئات اتش تحمل إما azidohomoalanine (آها، مركب ميثيونين) أو السكر غير طبيعي O-N-مرتبط azidoacetylglucosamine (O-GlcNAz)، وكلاهما والتي تحتوي على أزيد (N-3) وظيفية المجموعة. بعد تنقية للجزيئات الفيروس أزيد المعدلة، وligated تحقيقا آلكاين يحتوي على شاردة طمرة فلوري بطريقة chemoselective إلى البروتينات قبل المسمى أو بروتينات سكرية. أخيرا، يجري تحليل SDS-PAGE للتدليل على الربط الناجح للمسبار على البروتينات قفيصة الفيروسية. ويرد آها التأسيس لتسمية جميع قفيصة الفيروسيةالبروتينات (Hexon، بينتون والألياف)، في حين أن نتائج دمج O-GlcNAz في وضع العلامات من الألياف فقط. في هذا المجال المتطور، وقد تم طرق متعددة لأزيد-آلكاين ربط نجحت في تطوير، ولكن فقط وهما أننا وجدنا أن يكون الأكثر ملاءمة وأثبت هنا - السلالة التي تروج لها أزيد-آلكاين الاضافه الحلقيه (SPAAC) والنحاس والمحفز الاضافه الحلقيه أزيد-آلكاين (CuAAC) تحت جو امؤكسج.

Watch this Scientific Journal Video about Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry at JoVE.com

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

صممت جزيئات الفيروس الغدي لاحتواء إما غير طبيعي التماثلية الأحماض الأمينية azidohomoalanine أو السكر azido<em&gt; O</em&gt;-GlcNAz. وligated chemoselectively المجموعة أزيد من كل ردود الفعل عن طريق &quot;فوق&quot; كيمياء كوسيلة لتعديل سطح الفيروسية.

Chemoselective تعديل السطوح الفيروسية عن طريق الكيمياء نقرة Bioorthogonal

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

chemoselective-modification-viral-surfaces-via-bioorthogonal-click

Research

JoVE Journal

Immunology and Infection

23.8K Views.  Stony Brook University. صممت جزيئات الفيروس الغدي لاحتواء إما غير طبيعي التماثلية الأحماض الأمينية azidohomoalanine أو السكر azido O-GlcNAz. وligated chemoselectively المجموعة أزيد من كل ردود الفعل عن طريق "فوق" كيمياء كوسيلة لتعديل سطح الفيروسية.

Video: Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

باستخدام الفحص ميكروأري عموم الفيروسي (Virochip) إلى الشاشة العينات السريرية لمسببات الأمراض الفيروسية

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

بمعزل عن المتغيرات الاخلاص للفيروسات الحمض النووي الريبي وتوصيف التردد يتحور الفيروس

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

إعداد الحمض النووي الفيروسي من Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac تعديل النظام الأساسي للخلايا ينقول T الإنسان

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

إرساء ثقافة السائل المغطاة من مجرى الهواء الإنسان المستقطب طلائي Calu-3 خلايا لدراسة استجابة الخلية المضيفة لمسببات الأمراض التنفسية<em&gt; في المختبر</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

باستخدام الكيمياء فوق لقياس تأثير عدوى فيروسية في خلية المضيف RNA التجميعي

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

تطوير بوساطة حلقة-المجففة بالتبريد متساوي التضخيم الكواشف للكشف عن<em&gt; الكوكسيلا البورنيتية</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

قياس الأنفلونزا النورامينيداز تثبيط الجسم المضاد التتر من قبل انزيم مرتبط كتين الفحص

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

مسحة طريقة أخذ العينات للكشف عن نوروفيروس الإنسان على السطوح

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

ل<em&gt; في المختبر</em&gt; كاسيم ملزمة الفحص الذي يتنبأ اختراق المخدرات في الآفات السل

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...