וייטהד במכון עמיתי קרנות

חלק 1: הגדרת זיהום <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לגדל תאים הלה ב צלוחיות ולהמתין עד התאים הם כ 80% ומחוברות. </li><li style=";text-align:right;direction:rtl"> הכינו מספיק בינוני צמיחה ויראלי לניסוי: DMEM קבוע עם 2% FBS ללא אנטיביוטיקה הוסיף. </li><li style=";text-align:right;direction:rtl"> הזיהום יכול להתבצע גם עם מלאי גולמי של הנגיף vaccinia עם כייל ידוע, או סוכרוז מטוהרים וירוס עם כייל ידוע אם אתה מודאג לגבי ביטוי גנים המארח. </li><li style=";text-align:right;direction:rtl"> אם אתה משתמש וירוס סוכרוז מטוהרים, להמשיך ישירות חלק 2. </li><li style=";text-align:right;direction:rtl"> אם אתה משתמש מלאי וירוס גולמי vaccinia, זמן קצר לפני הזיהום, sonicate aliquot של הנגיף sonicator כוס עם אמבטיה של קרח בעיקר מעט מים. <ul style="list-style-type:lower-alpha;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Sonicate בסביבות 30W למשך 20 שניות. </li><li style=";text-align:right;direction:rtl"> וורטקס הצינור וחזור על sonication 3 פעמים, הוספת קרח יותר אם יש צורך לשמור על כוס מלא קרח צונן. </li><li style=";text-align:right;direction:rtl"> וורטקס בין כל שלב sonication. </li><li style=";text-align:right;direction:rtl"> המשך חלק 2. </li></ul></li><li style=";text-align:right;direction:rtl"> לחלופין, אם אינכם בעלי sonicator כוס, לערבב נפח שווה של המניה וירוס גולמי טריפסין 0.25mg/mL. <ul style="list-style-type:lower-alpha;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> וורטקס במרץ. </li><li style=";text-align:right;direction:rtl"> דגירה המניות / טריפסין וירוס לערבב 37 ° C waterbath במשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> וורטקס במרווחים 50-10 דקות. </li><li style=";text-align:right;direction:rtl"> המשך חלק 2. </li></ul></li></ol> חלק 2: הדבקת תאים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחשב את הסכום של חלקיקים נגיפיים צריך להדביק את monolayer על ריבוי הרצוי של זיהום (משרד הפנים). בדרך כלל, גבוה הפנים (בין 5 ל 10) משמש timecourses זיהום. </li><li style=";text-align:right;direction:rtl"> מוסיפים את כמות הרצויה של וירוס סוכרוז מטוהרים או וירוס גולמי trypsinized על 37 מעלות צלזיוס צמיחה התקשורת ויראלי ומערבבים היטב. אתה צריך להשתמש בתקשורת מספיק כדי לכסות רק את החלק התחתון של הבקבוק. לדוגמה, 10 מ&quot;ל של המדיה עבור בקבוק T-175. </li><li style=";text-align:right;direction:rtl"> הסר מדיה מן התאים ולשטוף עם בטמפרטורת החדר PBS. </li><li style=";text-align:right;direction:rtl"> הוסף את הוירוס / מדיה צמיחה ויראלי בקבוק אחד. <ul style="list-style-type:lower-alpha;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מערבולת בעדינות, צלחות הטיה לדגור על 37 מעלות צלזיוס ב 5% CO2 בחממה במשך שעה 1. </li><li style=";text-align:right;direction:rtl"> צלחות הטיה מערבולת כל 15 דקות כדי להפיץ וירוס אחיד לשמור תאים לחים. </li><li style=";text-align:right;direction:rtl"> עבור נקודת זמן 0hr/Mock, מוסיפים את התקשורת צמיחה ויראלי בלבד, עם וירוס לא הוסיף. </li></ul></li><li style=";text-align:right;direction:rtl"> לאחר דגירה 1 שעה, להסיר את המדיה המכילים את הנגיף, ולשטוף שלוש פעמים עם בטמפרטורת החדר PBS. </li><li style=";text-align:right;direction:rtl"> הוסף את הסכום המקסימלי של מדיום הגידול ויראלי בקבוק אחד. לדוגמה, 30 מ&quot;ל של המדיה עבור בקבוק T-175. מתחילים לספור את זה כנקודת 0 hr שלך זמן. </li></ol> חלק 3: תאים קציר <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בדוק את התאים תחת מיקרוסקופ לב כל השפעה cytopathic (CPE). </li><li style=";text-align:right;direction:rtl"> הסר את המדיה ולשטוף תאים עם 30 מ&quot;ל של בטמפרטורת החדר PBS. </li><li style=";text-align:right;direction:rtl"> הוסף 15 מ&quot;ל של טריפסין אל התאים דגירה עבור 2-5min על 37 ° C. </li><li style=";text-align:right;direction:rtl"> בדוק במיקרוסקופ על ניתוק התא ברז הבקבוק בעדינות כדי לסלק תאים. </li><li style=";text-align:right;direction:rtl"> העברת תאים עם טפטפת סרולוגיות סטרילי לתוך צינור חרוטי 50 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> לשטוף את הבקבוק עם נפח שווה של התקשורת צמיחת תאים, ולהוסיף את התקשורת התאים trypsinized בצינור חרוטי. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה התאים ב 300 x גרם במשך 5 דקות בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> הסר את טריפסין / מדיה מן התא גלולה. </li><li style=";text-align:right;direction:rtl"> Resuspend התא גלולה ב מגיב או TRIzol או החיץ תמוגה מתיק הרצוי RNA שלך בבידוד. </li><li style=";text-align:right;direction:rtl"> אם אתה משתמש TRIzol, לחלק את הדגימה לתוך 1 מ&quot;ל aliquots שכותרתו צינורות 1.5mL Eppendorf ולהקפיא ב -80 ° C. </li><li style=";text-align:right;direction:rtl"> חזור על הפעולה עבור כל נקודות הזמן, קצירת one בקבוק לכל נקודת זמן. </li></ol> חלק 4: מיצוי RNA של דגימות TRIzol <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בשלב זה, דגימות שלך צריך להיות resuspended ב מגיב TRIzol ומוכן להיות מעובד. </li><li style=";text-align:right;direction:rtl"> הוסף 200μL של מגיב הפרדת פאזות BCP עבור כל 1 מ&quot;ל של TRIzol בצינור אחד. ייתכן שיהיה עליך להעביר את הצינור מדגם גדול יותר אם אתה מתחיל עם כמות גדולה של TRIzol. </li><li style=";text-align:right;direction:rtl"> וורטקס תנער במרץ דגירה בטמפרטורת החדר למשך 2-3 דקות. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה מדגם XG ב 12,000 במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> מעבירים את השלב מימית לצינור טריים. השלב מימית היא שכבת ברור למעלה. אתה צריך להיות מחלים 600μL כ 1mL עבור כל TRIzol שהתחלת לצאת עם. </li><li style=";text-align:right;direction:rtl"> האם החילוץ פנול / כלורופורם שנייה, הפעם באמצעות 500μL של כלורופורם לכל 1 מ&quot;ל TRIzol, במקום BCP. </li><li style=";text-align:right;direction:rtl"> וורטקס תנער במרץ דגירה בטמפרטורת החדר למשך 2-3 דקות. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה מדגם XG ב 12,000 במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> מעבירים את השלב מימית לצינור טריים. השלב מימית היא שכבת ברור למעלה. </li><li style=";text-align:right;direction:rtl"> הוסף 20μg של acrylamide ליניארית מדגם זה. Acrylamide ליניארי מעשים כנשא ועוזר לזרז את הרנ&quot;א. </li><li style=";text-align:right;direction:rtl"> הוספת 500 &amp; miקרו; L של isopropanol לכל 1 מ&quot;ל של TRIzol שהתחלת לצאת עם לדגום כל ומערבבים היטב. </li><li style=";text-align:right;direction:rtl"> דגירה דגימות בטמפרטורת החדר למשך 10 דקות. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה ב microcentrifuge במהירות המרבית במשך 10-15 דקות. </li><li style=";text-align:right;direction:rtl"> גלולה RNA יוצג בחלק התחתון של הצינור. בזהירות רבה, להסיר את supernatant מהצינור, או עם aspirator ואקום, או באופן ידני על ידי pipettor. נא לא להפריע את הכדור. </li><li style=";text-align:right;direction:rtl"> שטפו את הכדור עם 1 מ&quot;ל של אתנול 70%. <ul style="list-style-type:lower-alpha;;text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף 1 מ&quot;ל של אתנול 70% על גלולה. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה שיא של 14,000 גרם במשך 5-7 דקות. </li><li style=";text-align:right;direction:rtl"> בזהירות רבה, להסיר את אתנול מן הצינור, או עם aspirator ואקום, או באופן ידני על ידי pipettor. בדוק כי גלולה גלוי בתחתית הצינור. </li><li style=";text-align:right;direction:rtl"> בטל supernatant. </li></ul></li><li style=";text-align:right;direction:rtl"> חזור על השלב לשטוף. לאחר supernatant נמחקת, לסמן היכן גלולה היא ברכבת התחתית. </li><li style=";text-align:right;direction:rtl"> האוויר יבש גלולה לא יותר מ 5 דקות. אל overdry, או RNA יהיה קשה לשחזר! </li><li style=";text-align:right;direction:rtl"> אם ברצונך לעקוב אחר הטיפול אופציונלי DNase להסיר כל שאר ה-DNA מן המדגם, resuspend גלולה ב 17μL מים nuclease חינם. אחרת, resuspend גלולה ב 20μL מים nuclease ללא והמשך לשלב 22. </li><li style=";text-align:right;direction:rtl"> (אופציונאלי טיפול DNase) באמצעות מערכת RNase ללא Qiagen DNase, להוסיף 2μL הצפת RDD ו 1μL מחדש RNase ללא DNase אני על RNA resuspended. מערבבים בעדינות. </li><li style=";text-align:right;direction:rtl"> (אופציונאלי טיפול DNase) לדגור על 37 מעלות צלזיוס במשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> (אופציונאלי טיפול DNase) הוסף 2μL של 2.5 mM EDTA ו לדגור על 65 מעלות צלזיוס למשך 5 דקות כדי להשבית DNase. אין לעבור את הזמן איון או טמפרטורה כמו פעמים יותר / טמפרטורות גבוהות יותר עלולות לגרום השפלה של רנ&quot;א. </li><li style=";text-align:right;direction:rtl"> בדוק את ריכוז RNA על ידי ספקטרופוטומטר. </li><li style=";text-align:right;direction:rtl"> חנות מדגם RNA ב -80 ° C. </li><li style=";text-align:right;direction:rtl"> (QC אופציונאלי) בדוק את איכות RNA באמצעות BioAnalyzer Agilent, או על ידי הפעלת המדגם על ג&#39;ל denaturing ובדיקת קריאות OD. (260nm ו - 260/280 יחס) </li></ol>

<ol>
	<li>KH, R. u. b. i. n. s., LE, H. e. n. s. l. e. y., GW, B. e. l. l., Wang, C., EJ, L. e. f. k. o. w. i. t. z., PO, B. r. o. w. n., DA, R. e. l. m. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+viral+gene+expression+programs+during+poxvirus+infection:+a+transcriptional+map+of+the+vaccinia+and+monkeypox+genomes.">Comparative analysis of viral gene expression programs during poxvirus infection: a transcriptional map of the vaccinia and monkeypox genomes.</a> PLoS ONE. 3 (7), e2628-e2628 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+a+complete+poxvirus+transcriptome+reveals+an+immediate-early+class+of+genes.">Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes.</a> Proc. Natl. Acad. Sci. 105 (6), 2140-2145 (2008).</li><li>Satheshkumar, P. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poxvirus+transcriptome+analysis.">Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E62-E62 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A.+Reply+to+Satheshkumar+and+Moss:+Poxvirus+transcriptome+analysis.">A. Reply to Satheshkumar and Moss: Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E63-E64 (2008).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., Mu&#241;oz, M., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+gene+expression+survey+of+vaccinia+virus+infection+of+human+HeLa+cells.">Cellular gene expression survey of vaccinia virus infection of human HeLa cells.</a> J Virol. 77 (11), 6493-6506 (2003).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Conde, R., Pascual-Montano, A., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+reveals+characteristic+changes+of+host+cell+gene+expression+in+response+to+attenuated+modified+vaccinia+virus+Ankara+infection+of+human+HeLa+cells.">Microarray analysis reveals characteristic changes of host cell gene expression in response to attenuated modified vaccinia virus Ankara infection of human HeLa cells.</a> J Virol. 78 (11), 5820-5824 (2004).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., N&#225;jera, J. L., Zaballos, A., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+response+to+the+attenuated+poxvirus+vector+NYVAC:+upregulation+of+apoptotic+genes+and+NF-kappaB-responsive+genes+in+infected+HeLa+cells.">Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.</a> J Virol. 80 (2), 985-998 (2006).</li><li>Guerra, S., N&#225;jera, J. L., Gonz&#225;lez, J. M., L&#243;pez-Fern&#225;ndez, L. A., Climent, N., JM, G. a. t. e. l. l., Gallart, T., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+gene+expression+profiling+after+infection+of+immature+human+monocyte-derived+dendritic+cells+by+the+attenuated+poxvirus+vectors+MVA+and+NYVAC.">Distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors MVA and NYVAC.</a> 81 (16), 8707-8721 (2007).</li></ol>

קריטי שלבים חלק 1 &amp; 2 ישנם שלבים קריטיים כמה הגדרת זיהום סינכרוני vaccinia, sonication first להיות זהיר (או trypzinizing) של הנגיף, על מנת disaggregate חלקיקי הנגיף. Vaccinia נוטה מאוד צבירה, לבין שיבוש של חלקיקי נגיף חשוב ל?...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>TRIzol Reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15596-026</td>
<td>Similar reagents, such as TriPure from Roche, will also work.</td>
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<td>BCP Phase Separation Reagent</td>
<td>Reagent</td>
<td>Molecular Research Center</td>
<td>BP151</td>
<td>&nbsp;</td>
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<td>RNase-Free DNase Set</td>
<td>Reagent</td>
<td>Qiagen</td>
<td>79254</td>
<td>DNase treatment is an optional step.</td>
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vaccinia virus infection &amp; temporal analysis of virus gene expression: part 1

המשפחה<em&gt; Poxviridae</em&gt; מורכב גדילי הדנ&quot;א הכפול גדולים המכילים וירוסים לשכפל אך ורק בציטופלסמה של תאים נגועים. חברי<em&gt; Orthopox</em&gt; הסוג כולל אבעבועות, הסוכן סיבתי של אבעבועות קטנות האדם, הקוף, ואת vaccinia (VAC), חבר prototypic משפחת וירוסים. בתוך גדולות יחסית (~ 200 kb) vaccinia הגנום, שלוש כיתות של גנים המקודדים: מוקדם, ביניים מאוחר. בעוד כל שלוש כיתות מתועתקים ידי virally בקידוד polymerases RNA, כל שיעור מהווה פונקציה שונה במחזור החיים של הנגיף. Poxviruses לנצל אסטרטגיות מרובים עבור אפנון של הסביבה המארחת הסלולר במהלך ההדבקה. כדי להבין את הרגולציה של המארח וגם גן ביטוי וירוס, יש לנו מנוצל הגנום כולו גישות לנתח שפע תמליל מנגיף וגם התאים המארחים. הנה, אנחנו מדגימים כמובן זמן זיהומים של תאים הלה בווירוס Vaccinia ו-RNA הדגימה במספר נקודות זמן לאחר הפגיעה. מארח שני ו-RNA ויראלי הכולל מופרד מוגבר של הכלאה כדי microarrays עבור ניתוח של ביטוי גנים.

Watch this Scientific Journal Video about Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1 at JoVE.com

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

פרוטוקול Vaccinia זיהום של תאים הלה וניתוח של המארח ביטוי גנים ויראליים. חלק 1 מתוך 3.

Vaccinia זיהום בנגיף וניתוח הזמני של ביטוי גנים וירוס: חלק 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

vaccinia-virus-infection-temporal-analysis-virus-gene-expression-part

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10.2K Views.  MIT - Massachusetts Institute of Technology. פרוטוקול Vaccinia זיהום של תאים הלה וניתוח של המארח ביטוי גנים ויראליים. חלק 1 מתוך 3.

Video: Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1

Bacterial Gene Expression Analysis Using Microarrays

Gene Expression חיידקית ניתוח שימוש microarrays

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

הכנת ראשי בתרביות תאים hematopoietic ממח העצם Murine עבור electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

Vaccinia זיהום בנגיף וניתוח הזמני של ביטוי גנים וירוס: חלק 2

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Vaccinia זיהום בנגיף וניתוח הזמני של ביטוי גנים וירוס: חלק 3

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Virus-Induced להשתקת גנים (VIGS) ב ניקוטיאנה benthamiana עגבניות

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

שימוש ג&#39;ין Pulser מערכת MXcell electroporation כדי Transfect תאים ראשוניים עם יעילות גבוהה

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

שימוש מונה תא אוטומטי כדי לפשט את הביטוי מחקרים ג&#39;ין: siRNA מציאה הביטוי IL 4-Gene תלויים תאים Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

ב הכינון במבחנה של ט פעילים castaneum שהטלומרז

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Agrobacterium בתיווך Virus-induced ג&#39;ין השתקת Assay כותנה

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

פלטפורמת הפקת MultiBac החלבון המורכבת בEMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...