ברצוננו להודות NSF למימון (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>

אין ניגוד עניינים הצהיר.

התפתחות של תגובות לחץ chemoselective ו bioorthogonal, כולל אלה של azides, הוא אזור מתפתח במהירות, מחקר, ולאחר מכן יש מספר גדל והולך של התגובות הללו לבחירה עבור יישומים bioconjugation. הגבלנו את היקף פרוטוקול זה כך שיכלול רק שתי שיטות אשר נבחרו בשל התועלת שלהם במעבדה שלנו וזמינות מסחרית של חומרים כימיים כל. במסלול הראשון מסוג זה - זן, לקדם אזיד-alkyne בחיי בן אשר (SPAAC) - alkyne נכלל בתוך טבעת cyclooctyne (איור 2 א). שיטה זו פותחה לאחרונה ניתן לבצע תחת אווירה מחומצן וללא צורך זרז נחושת. 9,10 עד לאחרונה, חיסרון אחד של שיטה זו היא סינתזה מייגע וזמינות מסחרית מופחת של אלה alkynes מתוחים לעומת מסוף alkynes שימוש נחושת (אני)-catalyzed אזיד-alkyne בחיי בן אשר (CuAAC), עם זאת, ספרייה הולכת וגדלה של בדיקות אלה זמין כעת מסחרית (ראה טבלה של חומרים כימיים). מסיבות אלה, בחרנו SPAAC כשיטת הבחירה בפרוטוקול זה. מתואר גם בפרוטוקול זה הוא קשירת אזיד-Modified חלקיקים adenoviral באמצעות CuAAC באווירה deoxygenated באמצעות bathophenanthroline disulfonic הרכב: חומצה מלח (BDA) כסוכן chelating (איור 2b). שיטה זו נוחה היא זרז BDA גם זמינים מסחרית וזולה. יתר על כן, דיווח התשואות הגבוה ביותר הם בדרך כלל משיטות, וסיבוכים הנובעים הדור של מינים החמצן מגיב (ראה מחומצן CuAAC, להלן) פתרון נמנעים. 8 עם זאת, נחושת (אני) זרז ידוע ציטוטוקסיות, 11 ו ציוד מיוחד הדרוש כדי ליישם הליך זה עשוי להרתיע כמה קבוצות מלהשתמש בה. עם זאת, אנו מוצאים שיטה זו יעילה שימושיadenovirus שינוי. השיטה שימושית 3 של CuAAC פותחה לאחרונה לעבוד בתנאים אטמוספריים מחומצן (איור 2 ג). 8 שיטה זו נהנה מאותן הטבות כמו הגישה BDA-לטיפולי קלאציה אבל משתמש THPTA במקום כסוכן chelating כדי להכיל חמצן אטמוספרי ללא הרס חמצוני של המצע. חסרון אחד קטן של גישה זו היא חוסר הנוכחי של הזמינות המסחרית של THPTA, למרות סינתזות פשוטות של הסוכן chelating דווחו. 8 למרות שלא ביצעו בשיטה קשירת במערכת שלנו, THPTA / CuAAC יהיה צפוי לייצר דומה התוצאות BDA / CuAAC, עם נוחות הוסיף אווירה של סובלנות מחומצן. למרות בכוונה לפתח את פרוטוקול סביב חומרים זמינים מסחרית, אנחנו עדיין רוצים להכיר את יתרונות העלות וגמישות מוגברת של סינתזהazides ו בדיקות alkyne בשימוש במסמך זה. Azidohomoalanine יכול להיות מוכן בתוך זמן קצר של 3 ימים עבור משמעותית פחות ממקורות מסחריים. 6,12 מדווחים ההכנות alkyne מתוחים קשה יותר 11 אבל להרשות לעצמם חופש הבחירה בדיקה.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td></tr><tr><td> אדנווירוס מסוג 5 (Ad5) המכיל transgene GFP </td><td> BCBC </td><td> 391 </td></tr><tr><td> כליה עובריים אנושיים (HEK 293) תאים </td><td> ATCC </td><td> CRL-1573 </td></tr><tr><td> Dulbecco של שינוי הנשר בינוני (DMEM), גלוקוז גבוהות </td><td> Invitrogen </td><td> 11965-092 </td></tr><tr><td> Dulbecco של שינוי הנשר בינוני, לא מתיונין, לא ציסטאין (DMEM-Met /-Cys), גלוקוז גבוהות </td><td> Invitrogen </td><td> 21013-024 </td></tr><tr><td> בקר עגל סרום </td><td> Invitrogen </td><td> 16170-078 </td></tr><tr><td> פניצילין - סטרפטומיצין 100 × פתרון (עט סטרפטוקוקוס) </td><td> Invitrogen </td><td> 15140-122 </td></tr><tr><td> 0.5% טריפסין-EDTA (10 ×) </td><td&gt; Invitrogen </td><td> 15400-054 </td></tr><tr><td> תא 100 מ&quot;מ צלחת תרבות, רקמות תרבות קלקר שטופלו </td><td> BD פלקון </td><td> 353003 </td></tr><tr><td> תרבית תאים CO 2 באינקובטור </td><td></td><td></td></tr><tr><td> Hemocytometer </td><td></td><td></td></tr><tr><td> בטיחות ביולוגית הממשלה </td><td></td><td></td></tr><tr><td> מזרק מסנן סטרילית (0.2 מיקרומטר, אצטט תאית) </td><td> VWR </td><td> 28145-477 (NA), 514-0061 (אירופה) </td></tr><tr><td> אוואנטי צנטריפוגות י&quot;א </td><td> Beckman Coulter </td><td> 369001 </td></tr><tr><td> צינורות חרוטי (50 מ&quot;ל, סטרילי) </td><td> BD פלקון </td><td> 352098 </td></tr><tr><td> , צינור Thinwall, Ultra-Clear, 13.2 מ&quot;ל, 14 x 89 מ&quot;מ </td><td> Beckman </td><td> 344059 </td></tr><tr><td> Ultracentrifuge מצויד 41 SW ו SW 60 rotor </td><td> Beckman </td><td></td></tr><tr><td> Eppendorf Biopur Safe-נעל צינורות, 1.5 מ&quot;ל </td><td> Eppendorf </td><td> 0030 121.589 </td></tr><tr><td> סרכזת 5418 </td><td> Eppendorf </td><td> 5418 </td></tr><tr><td> Centri בספט ספין ג&#39;ל סינון עמודות </td><td> פרינסטון הפרדות </td><td> CS-901 </td></tr><tr><td> מחט סטרילית, 18 מד </td><td></td><td></td></tr><tr><td> חנקן תיק הכפפה (אם CuAAC deoxygenated יש לבצעם) </td><td></td><td></td></tr><tr><td> בקבוק ויסקי </td><td></td><td></td></tr><tr><td> חנקן נוזלי </td><td></td><td></td></tr><tr><td> אלקטרופורזה התא </td><td></td><td></td></tr><tr><td> ג&#39;ל פלורסנט סורק </td><td></td><td></td></tr><tr><td> מוכן ג&#39;ל טריס-HCl ג&#39;ל </td><td> ביו רד </td><td> 161-1105 </td></tr><tr><td> L-Azidohomoalanine </td><td> AnaSpec </td><td> 63669 </td></tr><tr><td> Jena Biosciences </td><td> CLK-AA005 </td><td></td></tr><tr><td> Peracetylated N-azidoacetylgalactosamine (AC 4 GalNAz) </td><td> Invitrogen </td><td> C33365 </td></tr><tr><td> Thermo Scientific </td><td> 88905 </td><td></td></tr><tr><td> Sigma-Aldrich </td><td> A7480 </td><td></td></tr><tr><td> Bathophenanthroline disulfonic חומצה (BDA) הרכב: מלח </td><td> מגה פיקסל Biomedicals </td><td> 0215011201 </td></tr><tr><td> Sulfoxide דימתיל (DMSO) </td><td></td><td></td></tr><tr><td> מתנול </td><td></td><td></td></tr><tr><td> טריס הבסיס (טריס (hydroxymethyl) aminomethane) </td><td></td><td></td></tr><tr><td> הרכב: פוספט (Na2 HPO 4) </td><td></td><td></td></tr><tr><td> פוספט בופר סליין (PBS) </td><td></td><td></td></tr><tr><td> 1M HCl </td><td></td><td></td></tr><tr><td> גליצרול </td><td></td><td></td></tr><tr><td> שור אלבומין בסרום </td><td></td><td></td></tr><tr><td> L-ציסטאין </td><td></td><td></td></tr><tr><td> L-מתיונין </td><td></td><td></td></tr><tr><td> SDS (dodecyl נתרן גופרתי) </td><td></td><td></td></tr><tr><td> 2-mercaptoethanol </td><td></td><td></td></tr><tr><td> גליצין </td><td></td><td></td></tr><tr><td> Bromophenol כחול </td><td></td><td></td></tr><tr><td> כלוריד צסיום (CsCl) </td><td></td><td> &amp; Nbsp; </td></tr><tr><td> (אני) נחושת ברומיד (CuBr) </td><td></td><td></td></tr><tr><td> סידן כלוריד (CaCl 2) </td><td></td><td></td></tr><tr><td> אשלגן כלורי (KCl) </td><td></td><td></td></tr><tr><td> מגנזיום כלוריד (MgCl 2) </td><td></td><td></td></tr><tr><td> נתרן כלוריד (NaCl) </td><td></td><td></td></tr><tr><td colspan="3"></td></tr><tr><td> Alkyne בדיקה עבור * CuAAC </td><td></td><td></td></tr><tr><td> טמרה alkyne </td><td> Invitrogen </td><td> T10183 </td></tr><tr><td colspan="3"></td></tr><tr><td> בדיקה מאומצת alkyne עבור * SPAAC </td><td></td><td></td></tr><tr><td> טמרה DIBO alkyne </td><td> Invitrogen </td><td> C10410 </td></tr></tbody></table> * Quick הספקים הבולטים של כימיה לחץ ריאגנטים וערכות כוללים Invitrogen, Jena Biosciences, ברי Associates, Sigma-Aldrich, גלן מחקר, לחץ על כלים כימיה Baseclick. מגוון של צבעים alkyne ו ligands המיקוד ניתן למצוא בקטלוגים של אותם ספקים.

chemoselective modification of viral surfaces via bioorthogonal click chemistry

שינוי של חלקיקי נגיף קיבל כמות משמעותית של תשומת לב פוטנציאל אדיר שלו להשפיע על ריפוי גנטי, יישומים oncolytic ופיתוח החיסון. 1,2,3 גישות שוטפות למשקי שינוי משטחים ויראלי, שהם בעיקר גנטיקה מבוסס, לעיתים קרובות סובלים הנחתה התמרה של ייצור וירוס, infectivity ו הסלולר. 4,5 באמצעות כימיה לחץ chemoselective, פיתחנו גישה חלופית פשוטה אשר עוקפת בעיות אלה תוך שמירה הן גמישות מאוד נגיש. 1,2 מטרת פרוטוקול זה היא להדגים את היעילות של שימוש כימיה לחץ bioorthogonal לשנות את פני השטח של סוג adenovirus 5 חלקיקים. תהליך זה שני שלבים ניתן להשתמש גם טיפולית 1 או אנליטי, 2,6 כפי שהוא מאפשר קשירת chemoselective של מולקולות, צבעים מיקוד או מולקולות אחרות של עניין על חלבוניםמראש תווית עם תגי אזיד. שלושת היתרונות העיקריים של השיטה הם (1) תיוג מטבולית מדגים מעט השפעה על כושר ויראלי, 1,7 (2) מגוון רחב של ligands מפעיל יכול להיות מנוצל, ו (3) הוא מהיר להפליא, אמינות קל לגשת. 1,2,7 בשלב הראשון של הליך זה, חלקיקים adenovirus מיוצרים הנושא או azidohomoalanine (אהה, פונדקאית מתיונין) או סוכר טבעי O-linked N-azidoacetylglucosamine (O-GlcNAz), ושניהם מכילים אזיד (N-3) פונקציונלית הקבוצה. לאחר טיהור של אזיד-Modified חלקיקי נגיף, בדיקה alkyne המכיל מחצית ניאון טמרה הוא ligated באופן chemoselective על חלבונים או גליקופרוטאינים מראש שכותרתו. לבסוף, ניתוח SDS-PAGE מתבצע להפגין קשירת מוצלח של החללית על גבי חלבונים נגיפיים קפסיד. שילוב אהה מוצג לתייג כל capsid ויראליחלבונים (Hexon, פנטון ו Fiber), בזמן O-GlcNAz תוצאות לשילוב תיוג של סיבים בלבד. בתחום זה מתפתחת, מספר שיטות קשירת אזיד-alkyne פותחו בהצלחה, אך רק 2 מצאנו להיות הנוחה ביותר הם הפגינו להלן - זן, לקדם אזיד-alkyne בחיי בן אשר (SPAAC) ונחושת-catalyzed אזיד-alkyne בחיי בן אשר (CuAAC) תחת אווירה deoxygenated.

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

חלקיקים adenovirus מתוכננים להכיל אנלוגי טבעי חומצת אמינו azidohomoalanine או סוכר azido<em&gt; O</em&gt;-GlcNAz. קבוצה אזיד של כל אחד ligated chemoselectively דרך &quot;לחץ&quot; התגובות כימיה כאמצעי שינוי פני ויראלי.

שינוי Chemoselective של משטחים ויראלי באמצעות כימיה לחץ 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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

24.0K Views.  Stony Brook University. חלקיקים adenovirus מתוכננים להכיל אנלוגי טבעי חומצת אמינו azidohomoalanine או סוכר azido O-GlcNAz. קבוצה אזיד של כל אחד ligated chemoselectively דרך "לחץ" התגובות כימיה כאמצעי שינוי פני ויראלי.

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.

באמצעות assay הפאן ויראלי microarray (Virochip) אל מסך דוגמאות קליניות עבור פתוגנים ויראליים

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.

בידוד של פידליטי גרסאות של וירוסים RNA ו אפיון תדירות המוטציה וירוס

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.

ההכנה של ה-DNA ויראלי של Nucleocapsids

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

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

ייצור Titering של Adeno הקשורים ווקטורים רקומביננטי ויראלי

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

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.

שינוי מערכת Transposon piggyBac של תאי T אנושיים ראשוניים

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

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.

שימוש בכימיה לחץ כדי למדוד את ההשפעה של זיהום ויראלי בסינתזת RNA Host-Cell

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.

התפתחות Lyophilized Loop בתיווך ריאגנטים הגברה איזו תרמים לצורך זיהוי של<em&gt; Burnetii Coxiella</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.

מדידת טיטר נוגדנים עיכוב Neuraminidase שפעת ידי enzyme-linked Assay לקטינים

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.

שיטת הדגימה ספוגית לצורך זיהוי של norovirus האדם על משטחים

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.

An<em&gt; במבחנה</em&gt; Assay מחייב Assay כי חוזה חדירה לסמים בשחפת נגעים

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