עבודה זו נתמכת על ידי המכונים הלאומיים לבריאות (NIH R01 AI46435) ואת RMRCE (NIH AI065357).

<ol>
	<li>Brault, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+patterns+of+o'nyong+nyong+virus+in+the+malaria-transmitting+mosquito+Anopheles+gambiae.">Infection patterns of o'nyong nyong virus in the malaria-transmitting mosquito Anopheles gambiae.</a> Insect Mol. Biol. 13, 625-625 (2004).</li><li>Campbell, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aedes+aegypti+uses+RNA+interference+in+defense+against+Sindbis+virus+infection.">Aedes aegypti uses RNA interference in defense against Sindbis virus infection.</a> BMC. Microbiol. 8, 47-47 (2008).</li><li>Cirimotich, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+RNA+interference+increases+alphavirus+replication+and+virus-associated+mortality+in+Aedes+aegypti+mosquitoes.">Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes.</a> BMC. Microbiol. 9, 49-49 (2009).</li><li>Capurro, d. e. L. a. r. a., M, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-expressed,+recombinant+single-chain+antibody+blocks+sporozoite+infection+of+salivary+glands+in+Plasmodium+gallinaceum-infected+Aedes+aegypti.">Virus-expressed, recombinant single-chain antibody blocks sporozoite infection of salivary glands in Plasmodium gallinaceum-infected Aedes aegypti.</a> Am. J. Trop. Med. Hyg. 62, 427-427 (2000).</li><li>Foy, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+new+Sindbis+virus+transducing+system+and+its+characterization+in+three+Culicine+mosquitoes+and+two+lepidopteran+species.">Development of a new Sindbis virus transducing system and its characterization in three Culicine mosquitoes and two lepidopteran species.</a> Insect Mol. Biol. 13, 89-89 (2004).</li><li>Foy, B. D., Olson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alphavirus+transducing+systems.">Alphavirus transducing systems.</a> Adv. Exp. Med. Biol. 627, 19-19 (2008).</li><li>Franz, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+RNA+interference-based+resistance+to+dengue+virus+type+2+in+genetically+modified+Aedes+aegypti.">Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti.</a> Proc. Natl. Acad. Sci. U. S. A. 103, 4198-4198 (2006).</li><li>Hahn, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+Sindbis+virus+transient+expression+vectors+for+studying+antigen+processing+and+presentation.">Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation.</a> Proc. Natl. Acad. Sci. U. S. A. 89, 2679-2679 (1992).</li><li>Keene, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+acts+as+a+natural+antiviral+response+to+O'nyong-nyong+virus+(Alphavirus;+Togaviridae)+infection+of+Anopheles+gambiae.">RNA interference acts as a natural antiviral response to O'nyong-nyong virus (Alphavirus; Togaviridae) infection of Anopheles gambiae.</a> Proc. Natl. Acad. Sci. U. S. A. 101, 17240-17240 (2004).</li><li>Lundstrom, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+mammalian+membrane+proteins+in+mammalian+cells+using+Semliki+Forest+virus+vectors.">Expression of mammalian membrane proteins in mammalian cells using Semliki Forest virus vectors.</a> Methods Mol. Biol. 601, 149-149 (2010).</li><li>Myles, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alphavirus-derived+small+RNAs+modulate+pathogenesis+in+disease+vector+mosquitoes.">Alphavirus-derived small RNAs modulate pathogenesis in disease vector mosquitoes.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 19938-19938 (1993).</li><li>Olson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+resistance+to+dengue-2+virus+transmission+in+mosquitoes.">Genetically engineered resistance to dengue-2 virus transmission in mosquitoes.</a> Science. 272, 884-884 (1996).</li><li>Olson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+chloramphenicol+acetyltransferase+in+Aedes+albopictus+(C6/36)+cells+and+Aedes+triseriatus+mosquitoes+using+a+double+subgenomic+recombinant+Sindbis+virus.">The expression of chloramphenicol acetyltransferase in Aedes albopictus (C6/36) cells and Aedes triseriatus mosquitoes using a double subgenomic recombinant Sindbis virus.</a> Insect Biochem. Mol. Biol. 24, 39-39 (1994).</li><li>Olson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Sindbis+virus+expression+system+that+efficiently+expresses+green+fluorescent+protein+in+midguts+of+Aedes+aegypti+following+per+os+infection.">Development of a Sindbis virus expression system that efficiently expresses green fluorescent protein in midguts of Aedes aegypti following per os infection.</a> Insect Mol. Biol. 9, 57-57 (2000).</li><li>Parikh, G. R., Oliver, J. D., Bartholomay, L. C., C, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+haemocyte+tropism+for+an+arbovirus.">A haemocyte tropism for an arbovirus.</a> J. Gen. Virol. 90, 292-292 (2009).</li><li>Pierro, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+orally+infectious+Sindbis+virus+transducing+system+that+efficiently+disseminates+and+expresses+green+fluorescent+protein+in+Aedes+aegypti.">Development of an orally infectious Sindbis virus transducing system that efficiently disseminates and expresses green fluorescent protein in Aedes aegypti.</a> Insect Mol. Biol. 12, 107-107 (2003).</li><li>Rutledge, L. C. W. a. r. d., A, R., Gould, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+feeding+response+of+mosquitoes+to+nutritive+solutions+in+a+new+membrane+feeder.">Studies on the feeding response of mosquitoes to nutritive solutions in a new membrane feeder.</a> Mosq. News. 24, 407-407 (1964).</li><li>Sanders, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sindbis+virus+induces+transport+processes+and+alters+expression+of+innate+immunity+pathway+genes+in+the+midgut+of+the+disease+vector+Aedes+aegypti.">Sindbis virus induces transport processes and alters expression of innate immunity pathway genes in the midgut of the disease vector Aedes aegypti.</a> Insect Biochem. Mol. Biol. 35, 1293-1293 (2005).</li><li>Uhlirova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Sindbis+virus-mediated+RNA+interference+to+demonstrate+a+conserved+role+of+Broad-Complex+in+insect+metamorphosis.">Use of Sindbis virus-mediated RNA interference to demonstrate a conserved role of Broad-Complex in insect metamorphosis.</a> Proc. Natl. Acad. Sci. U. S. A. 100, 15607-15607 (2003).</li><li>Vanlandingham, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+double+subgenomic+chikungunya+virus+infectious+clone+to+express+heterologous+genes+in+Aedes+aegypti+mosquitoes.">Development and characterization of a double subgenomic chikungunya virus infectious clone to express heterologous genes in Aedes aegypti mosquitoes.</a> Insect Biochem. Mol. Biol. 35, 1162-1162 (2005).</li><li>Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+inducing+or+inhibiting+apoptosis+on+Sindbis+virus+replication+in+mosquito+cells.">Effects of inducing or inhibiting apoptosis on Sindbis virus replication in mosquito cells.</a> J. Gen. Virol. 89, 2651-2651 (2008).</li></ol>

אין ניגודי אינטרסים הכריז.

השיטות שהוצגו כאן לאפשר לחוקרים לעקוב אחר זיהום של alphavirus בתרבות התא יתושים היתוש נקבה בוגרת. היתרונות והחסרונות של השימוש וירוסים ATS מסוכמים בטבלה 2. שיבוט ATS זיהומיות ניתן להשפיע גנטית כדי לבטא כל GOI ולא היה בהם כדי להביע נוגדנים שרשרת אחת, מדכאי של RNAi, RNAs antisense מיקוד גנים אנדוגניים, ופרו חלבונים אפופטוטיים. אם עיתונאי אינו בשימוש, הרי חלק הדמיה של שיטה זו אינה רלוונטית. עם זאת, רבים של הפרוטוקולים זהה הכרחיים להצלת וירוס ATS, התפשטות, זיהום יתושים. ישנן מגבלות בגודל מקסימלי של transgene כאשר מוכנס לתוך מערכת transducing alphavirus. מגבלות גודל משתנים בהתאם לזן ההורים השתמשו כדי ליצור את ATS, אבל הם בדרך כלל על 1kb למרות הגנים הכתב גדול כמו גחלילית בלוציפראז שימשו בבניית ATS לשימוש יתושים. מעבדות מחקר עם סכום צנוע של רקע וירולוגיה אמור להיות מסוגל להשתמש ATS הבא של הפרוטוקולים הללו. מספר מעבדות מחקר עשית זאת באמצעות SINV מבוססי של ATS.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>QIAprep Spin Miniprep Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>27104</td>
<td>&nbsp;</td>
<tr>
<td>MAXIscript Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AB1312</td>
<td>Be sure to use a kit containing the appropriate polymerase for your construct.</td>
</tr>
<tr>
<td>Cap Analogue (m7G(5')ppp(5')G)</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM8050</td>
<td>&nbsp;</td>
<tr>
<td>Nanoject II nanoliter injector</td>
<td>&nbsp;</td>
<td>Drummond Scientific</td>
<td>3-000-204</td>
<td>&nbsp;</td>
<tr>
<td>Electro Cell Manipulator</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>ECM 630</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

alphavirus transducing system:  tools for visualizing infection in mosquito vectors

Alphavirus מערכות transducing (ATSs) הם כלי חשוב לביטוי גנים עניין (GOI) במהלך ההדבקה. ATSs נגזרות שיבוטים cDNA של יתושים וירוסים RNA (Alphavirus הסוג; משפחה Togaviridae). הסוג Alphavirus מכיל כ 30 שונים יתושים מינים וירוס. Alphaviruses הם נגיפים עטופים ומכילים חד גדילי RNA בגנום (~ 11.7 Kb). Alphaviruses לתמלל subgenomic mRNA המקודד חלבונים מבניים של הנגיף נדרש encapsidation של הגנום והתבגרות של הנגיף. Alphaviruses הם בדרך כלל ממס ביותר בתאים בעלי חוליות, אך בהתמדה להדביק תאים רגישים יתוש עם cytopathology מינימלית. תכונות אלו הופכות אותם כלי מעולה עבור ביטוי גנים וקטורים יתושים. ATSs הנפוץ ביותר בשימוש נגזרות Sindbis וירוס (SINV). כמיהות מינים רחב של SINV מאפשר הדבקה של cells8 חרקים, העופות, היונקים. עם זאת, ATSs כבר נגזר alphaviruses אחרים, כמו גם 9,10,20. ביטוי גנים זרים מתאפשרת על ידי החדרה של אתר נוסף נגיפי RNA חניכה subgenomic או האמרגן. ATSs שבו רצף הגן אקסוגניים ממוקם 5 &quot;לגנים מבניים ויראלי משמש ביטוי חלבון יציב חרקים. ATSs, שבו רצף הגן ממוקם 3 &quot;לגנים מבניים, משמש להפעלת RNAi ושתיקה הביטוי של הגן כי החרק. ATSs הוכיחו להיות כלי חשוב להבנת וקטור הפתוגן אינטראקציות, פרטים המולקולרי של שכפול נגיפי מחזורי תחזוקה זיהומיות 3,4,11,19,21. בפרט, את הביטוי של עיתונאים פלורסנט bioluminescent כבר סייעה מעקב אחר זיהום ויראלי בשידור וקטור הווירוס 5,14-16,18. בנוסף, התגובה החיסונית וקטור תוארה באמצעות שני זנים של SINV שהונדסו GFP 2,9. כאן, אנו מציגים שיטה לייצור SINV ובו כתב ניאון (GFP) מ שיבוט cDNA זיהומיות. זיהומיות, באורך מלא RNA הוא עיבד מ שיבוט cDNA לינארית. זיהומיות RNA מוחדר לתאים יעד מתירני ידי electroporation. תאים transfected ליצור חלקיקי נגיף מדבק המבטא את GOI. וירוס שנקטפו משמש להדביק יתושים, כפי שתואר כאן, או לארח מינים אחרים (לא מוצג כאן). יכולת וקטור נבחנת על ידי גילוי הקרינה מחוץ midgut או על ידי ניטור הנגיף 7 הילוכים. שימוש עיתונאי ניאון כמו GOI מאפשר אמידה נוח להתפשטות הנגיף ברחבי התרבות תאים, לצורך קביעת שיעור הפצת הזיהום, על יתושים חשוף, העברת הנגיף היתוש ומספק מד מהירה של יכולת וקטור.

Watch this Scientific Journal Video about Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors at JoVE.com

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

שיטות באמצעות מערכות alphavirus transducing להביע כתבים פלורסנט במבחנה יתושים בוגרים מתוארים. טכניקה זו עשויה להיות מותאם כדי להביע כל חלבון של עניין במקום או בנוסף לכתב.

מערכת Alphavirus Transducing: כלי המאפשר הדמיה זיהום ווקטורים יתוש

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

alphavirus-transducing-system-tools-for-visualizing-infection

Research

JoVE Journal

Immunology and Infection

14.7K Views.  Colorado State University. שיטות באמצעות מערכות alphavirus transducing להביע כתבים פלורסנט במבחנה יתושים בוגרים מתוארים. טכניקה זו עשויה להיות מותאם כדי להביע כל חלבון של עניין במקום או בנוסף לכתב.

Video: Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

פרוטוקול איסוף מכתים Hemocytes מן יתוש הקדחת הצהובה Aedes aegypti</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

Visualizing Dengue Virus through Alexa Fluor Labeling

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

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

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

לדמיין וירוס דנגה דרך אלקסה פלואוריד תיוג

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

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 ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia culturing ממערכת העצבים המרכזי וילודים למבוגרים

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

הפקת חלבון סה&quot;כ ו2-D שיטות ג&#39;ל אלקטרופורזה עבור<em&gt; Burkholderia</em&gt; מינים

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

גישה פשוטה ויעילה לבנות וקטורי וירוס Vaccinia Mutant

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

לדמיין את ההשפעות של כיח על פיתוח biofilm באמצעות מודל תאיים coverglass

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM.
The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.

This manuscript describes the use of transgenic reporter mice and different administration routes of fluorescent dyes in angiotensin II-induced hypertension using intravital video microscopy of blood vessels to evaluate the activation of immune cells and their ability to roll and adhere to the endothelium.

להמחיש ליקוציט מתגלגל והצמדות אנגיוטנסין II חדורים עכברים: טכניקות ואת החסרונות

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

יצירת וקטורים ההרפס העופות רקומביננטי עם CRISPR/Cas9 ג'ין עריכה

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

זיהום חוזר בדרכי השתן של Escherichia coli שנגרם על ידי גרדנרלה וגינליס חשיפה לשלפוחית השתן בעכברים

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...