עבודה זו נתמכה על ידי משרד המחקר של הצי הימי של ארה&quot;ב באמצעות כספים במעבדת המחקר הליבה.

<ol>
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Probes. 17, 165-169 (2003).</li><li>Obika, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+2'-O,+4'-C-methyleneuridine+and+-cytidine.+Novel+bicyclic+nucleosides+having+a+fixed+C3'-endo+sugar+puckering.">Synthesis of 2'-O, 4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3'-endo sugar puckering.</a> Tetrahedron. Lett. 38, 8735-8738 (1997).</li><li>Koshkin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LNA+(locked+nucleic+acids):+synthesis+of+the+adenine,+cytosine,+guanine,+5-methylcytosine,+thymine,+and+uracil+bicyclonucleoside+monomers+oligomerisation,+and+unprecedented+nucleic+acid+recognition.">LNA (locked nucleic acids): synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil bicyclonucleoside monomers oligomerisation, and unprecedented nucleic acid recognition.</a> Tetrahedron. 54, 3607-3630 (1998).</li><li>Kubota, K., Ohashi, A., Imachi, H., Harada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+in+situ+hybridization+efficiency+with+locked-nucleic-acid-incorporated+DNA+probes.">Improved in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes.</a> Appl. Environ. Microbiol. 72, 5311-5317 (2006).</li><li>Robertson, K. L., Thach, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LNA+flow-FISH:+A+flow+cytometry-fluorescence+in+situ+hybridization+method+to+detect+messenger+RNA+using+locked+nucleic+acid+probes.">LNA flow-FISH: A flow cytometry-fluorescence in situ hybridization method to detect messenger RNA using locked nucleic acid probes.</a> Anal. Biochem. 390, 109-114 (2009).</li><li>Waters, L., Storz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+RNAs+in+bacteria.">Regulatory RNAs in bacteria.</a> Cell. 136, 615-628 (2009).</li><li>Petersen, M., Wengel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LNA:+a+versatile+tool+for+therapeutics+and+genomics.">LNA: a versatile tool for therapeutics and genomics.</a> Trends. Biotechnol. 21, 74-81 (2003).</li><li>Corput, M. P. C. v. d., Grosveld, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+Situ+Hybridization+Analysis+of+Transcript+Dynamics+in+Cells.">Fluorescence in Situ Hybridization Analysis of Transcript Dynamics in Cells.</a> Methods. 25, 111-118 (2001).</li><li>Silveira, A. C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+non-coding+RNAs+in+environmental+vibrios.">Identification of non-coding RNAs in environmental vibrios.</a> Microbiology. 156, 2452-2458 (2010).</li></ol>

המחברים אין לחשוף.

השיטה זרימה FISH LNA המוצג כאן נעשה שימוש כדי לאתר את הביטוי של sRNA מן גראם שליליים ימיים החיידק Vibrio campbellii אשר הביטוי כבר בעבר אישרו דרך פרופיל microarray המבוסס על הביטוי שעתוק לאחור polymerase תגובת שרשרת 16. עד כה השתמשנו בשיטה זו כדי לעקוב אחר הביטוי של מגוון רחב של RNAs (למשל טרנס מקודד sRNA, sRNA כי לווסת את פעילות החלבון, riboswitches, ו-mRNA). ככזה, אנו בטוחים כי השיטה ניתנת להתאמה והוא יכול לשמש כדי לזהות כל יעד sRNA או mRNA והוא יכול להיות שונה לשימוש בכל מיני חיידקים או סוג תא. אם שינוי של פרוטוקול זה הוא הכרחי עבור סוגי תאים אחרים, המשתנה החשוב ביותר לשקול מניפולציה היא הצעד permeabilization. לדוגמה, כאשר שינוי תנאי permeabilization המתואר כאן, שינויים בריכוזים של ליזוזים ו proteinase K צריך להיבדק. מצאנו כי הכפלת אושילש את כמות ליזוזים ו proteinase K הביא שינויים משמעותיים הזרימה-FISH תוצאות LNA. יתר על כן, כאשר בדיקות permeabilization תנאים נוספים, התאים יש לנתח עבור clumping, אובדן התא האות השגה הטוב ביותר יחס רקע פלואורסצנטי. היקף clumping התא יכול להיבדק על ידי מיקרוסקופיה / או cytometry הזרימה. עד cytometry זרימה, גושים תא קיימים כמו זנבות של קדימה לעומת פיזור נקודה בצד העלילה ואת שיטת permeabilization הנכון יהיה למזער את האפקט הזה עוקב. שיטה זו היא גם מקובל זיהוי זמנית sRNA בלי שינויים גדולים לפרוטוקול כמתואר בדיקות נוספות LNA שכותרתו עם haptens אחרים כגון digoxigenin ניתן להוסיף במהלך שלב הכלאה ו זוהה אז עם נוגדן אנטי digoxigenin ו המצומד משני נוגדנים fluorophore. נכון לעכשיו, המגבלה היחידה שאנו נתקלים בשיטה זו היא חוסר יכולתה ליצור אות מספקת מצד לשעבר חלושותמינים sRNA נלחץ מאמצים נערכים כדי לקבוע את סף זיהוי (מספר העתק מוחלט לכל תא). בסך הכל, את שיטת תזרים LNA-FISH מספקת את ההזדמנות כדי למדוד sRNA חיידקי או ביטוי mRNA ברמת תא בודד באופן תפוקה גבוהה, כדי לספק תובנות על נוכחות השפע היחסי של מינים sRNA ואת מידת הביטוי שלו משתנה באוכלוסייה.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> פוספט 10X שנאגרו מלוחים (PBS), pH 7.4 </td><td> Applied Biosystems / Ambion </td><td> AM9625 </td><td></td></tr><tr><td> Nuclease ללא מים </td><td> Applied Biosystems / Ambion </td><td> AM9932 </td><td> (לא DEPC מטופלים) </td></tr><tr><td> 32% paraformaldehyde </td><td> מיקרוסקופית אלקטרונים למדעים </td><td> RT 15714 </td><td> אתנול חינם </td></tr><tr><td> חומצה אצטית </td><td> Acros אורגניקס </td><td> 327290010 </td><td></td></tr><tr><td> Diethyl pyrocarbonate (DEPC) </td><td> סיגמא אולדריץ&#39; </td><td> D5758 </td><tד&gt; שמור מיובש </td></tr><tr><td> ליזוזים </td><td> סיגמא אולדריץ&#39; </td><td> L2879 </td><td></td></tr><tr><td> Proteinase K (20 מ&quot;ג / מ&quot;ל) </td><td> Applied Biosystems / Ambion </td><td> AM2546 </td><td></td></tr><tr><td> לפוראמיד </td><td> Applied Biosystems / Ambion </td><td> AM9342 </td><td> Aliquot Deionized, ולהקפיא </td></tr><tr><td> Dextran סולפט MW&gt; 500.000 </td><td> סיגמא אולדריץ&#39; </td><td> D8906 </td><td> Aliquot פתרון 60% ולהקפיא </td></tr><tr><td> 50X פתרון Denhardt נתרכז </td><td> סיגמא אולדריץ&#39; </td><td> D2532 </td><td> Aliquot ולהקפיא </td></tr><tr><td> סודיום ציטרט חיץ 20X </td><td> Applied Biosystems / Ambion </td><td> AM9770 </td><td></td></tr><tr> &lt;td&gt; זרע DNA סלמון (טעון) 10 מ&quot;ג / מ&quot;ל </td><td> Applied Biosystems / Ambion </td><td> AM9680 </td><td> Aliquot ולהקפיא </td></tr><tr><td> שמרים tRNA (10 מ&quot;ג / מ&quot;ל) </td><td> Life Technologies / Invitrogen </td><td> 15401-011 </td><td> Aliquot ולהקפיא </td></tr><tr><td> Tween-20 </td><td> סיגמא אולדריץ&#39; </td><td> P9416 </td><td></td></tr><tr><td> 5X בפתרון הכלאה באתרו חסימת </td><td> וקטור מעבדות </td><td> MB-1220 </td><td></td></tr><tr><td> DyLight 488 streptavidin </td><td> וקטור מעבדות </td><td> SA-5488-1 </td><td></td></tr><tr><td> Biotinylated אנטי streptavidin </td><td> וקטור מעבדות </td><td> BA-0500 </td><td> Aliquot ולהקפיא </td></tr></tbody></table>

locked nucleic acid flow cytometry-fluorescence in situ hybridization (lna flow-fish): a method for bacterial small rna detection

הקרינה הכלאה באתרו (FISH) היא טכניקה רבת עוצמה המשמש לזהות בתרגום ספציפי רצפי חומצות גרעין בסביבה הסלולרית. על מנת להגדיל את התפוקה, דגים ניתן לשלב עם cytometry זרימה (flow-FISH) כדי לאפשר את זיהוי ממוקד של רצפי חומצות גרעין אלפי תאים בודדים. כתוצאה מכך, תזרים-FISH מציעה יתרון בולט על פני lysate / אנסמבל מבוססי חומצת גרעין לזיהוי שיטות כי כל תא היא כאל תצפית עצמאית, ובכך לאפשר ניתוח סטטיסטי ושונות חזק. תכונות אלה גרמו שימוש בשיטות דגים זרימה דגים במספר יישומים שונים בכלי של שיטות אלה הודגמה בהצלחה בזיהוי הטלומרים קביעת אורך, 1,2 הסלולר ביטוי גנים 3,4, ניטור כפל ויראלי תאים נגועים 5, וניתוח הקהילה ספירת חיידקים6. באופן מסורתי, סגוליות של שיטות דגים זרימה דגים כבר הנחילה ידי בדיקות דנ&quot;א oligonucleotide. ואולם, החלפת oligonucleotide בדיקות DNA עם אנלוגים של חומצות גרעין כמו בדיקות דגים זרימה FISH גדל הן רגישות וספציפיות של טכניקה זו בשל הטמפרטורות היתוך גבוהה (T מ &#39;) של אנלוגים אלו חומצות גרעין טבעי 7,8 . חומצות גרעין נעול (LNA) בדיקות הן סוג של חומצות גרעין אנלוגי המכילים נוקלאוטידים LNA זינק לאורך רצף ה-DNA או RNA 9,10. בשילוב עם דגים זרימה, בדיקות LNA בעבר הוכח להכות בדיקות דנ&quot;א קונבנציונלי 7,11 ו כבר השתמשו בהצלחה כדי לזהות mRNA אוקריוטים 12 ו-RNA נגיפי בתאי יונקים 5. כאן אנו להרחיב את יכולת לתאר שיטה זרימה FISH LNA אשר מאפשר זיהוי ספציפי של RNA בתא החיידקs (איור 1). באופן ספציפי, אנו מעוניינים זיהוי של קטן ללא קידוד הרגולציה RNA (sRNA) אשר משכו עניין רב בשנים האחרונות כפי שהם נמצאו לשמש גורמים רגולטוריים מרכזיים רבים תהליכים תאיים קריטי 13. עם זאת, יש כלים מוגבלים ללמוד sRNAs ואת האתגרים של גילוי sRNA בתאי החיידקים נובע בחלקו לגודל קטן יחסית (בדרך כלל 5-30 נוקלאוטידים אורך) ושפע נמוך של מולקולות sRNA כמו גם את הקושי הכללי בעבודה עם תאים ביולוגיים קטנים יותר עם משתנים ממברנות התאים. בשיטה זו, אנו מתארים קיבעון ו permeabilzation התנאים לשמר את המבנה של תאים חיידקיים ולאפשר את חדירת בדיקות LNA, כמו גם הגברה אות צעדים המאפשרים זיהוי ספציפי של שפע sRNA נמוך (איור 2).

Watch this Scientific Journal Video about Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection at JoVE.com

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

שיטה חדשנית תפוקה גבוהה מתואר המאפשר איתור quantitation היחסי של RNA קטנים ביטוי mRNA של תאים חיידקיים יחיד באמצעות נעול בדיקות חומצות גרעין תזרים cytometry פלואורסצנציה<em&gt; באתרו</emהכלאה&gt;.

נעול חומצות גרעין תזרים cytometry פלואורסצנציה באתרו</emהכלאה> (LNA זרימה FISH): שיטה לגילוי חיידקי RNA קטנים

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

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Immunology and Infection

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

25.2K Views.  Naval Research Laboratory. שיטה חדשנית תפוקה גבוהה מתואר המאפשר איתור quantitation היחסי של RNA קטנים ביטוי mRNA של תאים חיידקיים יחיד באמצעות נעול בדיקות חומצות גרעין תזרים cytometry פלואורסצנציה באתרו.

Video: Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

שילוב של הדגימה דבק קלטת מבוססי פלואורסצנטי באתרו הכלאה עבור איתור מהיר של סלמונלה על תוצרת טרייה

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development 1. From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in 2). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice 3,4. We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.

Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.

Transfection ו mutagenesis של גני מטרה של תאים על ידי יתוש נעול חומצה שונה oligonucleotides גרעין

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

&quot;Bioluminescent&quot; כתב phage לצורך זיהוי של קטגוריה פתוגנים חיידקיים

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

הכלאה<em&gt; באתרו</em&gt; של בלוטות הרוק, השחלות, ועוברים של וקטור יתושים

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

פלואורני<em&gt; באתר</em&gt; הכלאה על כרומוזומים mitotic של יתושים

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

חיידקים מלאכותיים כרומוזומים: Genomics כלי פונקציונלי לחקר חיובי גדיל RNA וירוסים

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

חילוץ מהיר, בטוח, ופשוט ידנית המיטה חומצת גרעין איתור וירוס בדגימות דם מלא

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

פלואורסצנטית כמותי באתרו היברידיזציה (דג) ו Immunofluorescence (IF) של מוצרים גנטיים ספציפיים בתאים שנדבקו KSHV

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

דוגמנות דלקת כבד B וירוס ב-t-הפטיטיס 293T-NE-3NRs תאים

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336&#916;micC by the &#955; Red-based recombination system and the complemented mutant 50336&#916;micC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336&#916;micC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336&#916;micC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336&#916;micC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336&#916;micC and 50336&#916;micC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 109 CFU, 1.55 x 108 CFU, and 2.54 x 108 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

An &#955;-Red-mediated recombination system was used to create a deletion mutant of a small non-coding RNA micC.

RNA קטן שאינו מקודד תורם לאלימות בחלבוני הממברנה החיצונית בסלמונלה אנטריטידיס

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia ...

נעול חומצות גרעין תזרים cytometry פלואורסצנציה באתרו (LNA זרימה FISH): שיטה לגילוי חיידקי RNA קטנים