אנו מודים ד&quot;ר רנדי אלקינס, רובין Kastenmayer, טד טורי, דן לגזור טובי ליהמן על קריאה ביקורתית של כתבי יד. עבודה זו נתמכה על ידי תוכנית המחקר החומות של חטיבת המחקר של בין החומות, המכון הלאומי לאלרגיה ומחלות זיהומיות, המכונים הלאומיים לבריאות, ועל ידי התוכנית הלאומית למחקר בסיסי 973 של סין, # 2007CB513103. אנו מודים NIAID עירונית עורך ברנדה ריי מרשל לסיוע.

<ol>
	<li>Hayton, K., Ranford-Cartwright, L. C., Walliker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfadoxine-pyrimethamine+resistance+in+the+rodent+malaria+parasite+Plasmodium+chabaudi.">Sulfadoxine-pyrimethamine resistance in the rodent malaria parasite Plasmodium chabaudi.</a> Antimicrob Agents Chemother. 46, 2482-2489 (2002).</li><li>Cravo, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+mefloquine+resistance+in+the+rodent+malaria+parasite+Plasmodium+chabaudi.">Genetics of mefloquine resistance in the rodent malaria parasite Plasmodium chabaudi.</a> Antimicrob Agents Chemother. 47, 709-718 (2003).</li><li>Hunt, P., Cravo, P. V., Donleavy, P., Carlton, J. M., Walliker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroquine+resistance+in+Plasmodium+chabaudi:+are+chloroquine-resistance+transporter+(crt)+and+multi-drug+resistance+(mdr1)+orthologues+involved?.">Chloroquine resistance in Plasmodium chabaudi: are chloroquine-resistance transporter (crt) and multi-drug resistance (mdr1) orthologues involved?.</a> Mol Biochem Parasitol. 133, 27-35 (2004).</li><li>Yuan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mapping+of+targets+mediating+differential+chemical+phenotypes+in+Plasmodium+falciparum.">Genetic mapping of targets mediating differential chemical phenotypes in Plasmodium falciparum.</a> Nat Chem Biol. 5, 765-771 (2009).</li><li>Su, X., Kirkman, L. A., Fujioka, H., Wellems, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+polymorphisms+in+an+approximately+330+kDa+protein+are+linked+to+chloroquine-resistant+P.+falciparum+in+Southeast+Asia+and+Africa.">Complex polymorphisms in an approximately 330 kDa protein are linked to chloroquine-resistant P. falciparum in Southeast Asia and Africa.</a> Cell. 91, 593-603 (1997).</li><li>Hayton, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte+binding+protein+PfRH5+polymorphisms+determine+species-specific+pathways+of+Plasmodium+falciparum+invasion.">Erythrocyte binding protein PfRH5 polymorphisms determine species-specific pathways of Plasmodium falciparum invasion.</a> Cell Host Microbe. 4, 40-51 (2008).</li><li>Pattaradilokrat, S., Cheesman, S. J., Carter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congenicity+and+genetic+polymorphism+in+cloned+lines+derived+from+a+single+isolate+of+a+rodent+malaria+parasite.">Congenicity and genetic polymorphism in cloned lines derived from a single isolate of a rodent malaria parasite.</a> Mol Biochem Parasitol. 157, 244-247 (2008).</li><li>Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hundreds+of+microsatellites+for+genotyping+Plasmodium+yoelii+parasites.">Hundreds of microsatellites for genotyping Plasmodium yoelii parasites.</a> Mol Biochem Parasitol. 166, 153-158 (2009).</li><li>Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Typing+Plasmodium+yoelii+microsatellites+using+a+simple+and+affordable+fluorescent+labeling+method.">Typing Plasmodium yoelii microsatellites using a simple and affordable fluorescent labeling method.</a> Mol Biochem Parasitol. 155, 94-102 (2007).</li><li>Walliker, D., Carter, R., Morgan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+recombination+in+malaria+parasites.">Genetic recombination in malaria parasites.</a> Nature. 232, 561-562 (1971).</li><li>Sinden, R. E., Hartley, R. H. <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+the+meiotic+division+of+malarial+parasites.">Identification of the meiotic division of malarial parasites.</a> J Protozool. 32, 742-744 (1985).</li><li>Ozaki, L. S., Gwadz, R. W., Godson, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+centrifugation+method+for+rapid+separation+of+sporozoites+from+mosquitoes.">Simple centrifugation method for rapid separation of sporozoites from mosquitoes.</a> J Parasitol. 70, 831-833 (1984).</li><li>Yoeli, M., Most, H., Bone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+berghei:+cyclical+transmission+by+experimentally+infected+Anopheles+quadrimachulatus.">Plasmodium berghei: cyclical transmission by experimentally infected Anopheles quadrimachulatus.</a> Science. 144, 1580-1581 (1964).</li><li>Landau, I., Boulard, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Life cycles and Morphology. , (1978).</li><li>Vanderbergh, J. P. Y., M, . <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+temperature+on+sporogonic+development+of+Plasmodium+berghei.">Effects of temperature on sporogonic development of Plasmodium berghei.</a> J Parasitol. 52, 559-564 (1966).</li><li>Gadsby, N., Lawrence, R., Carter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+on+pathogenicity+and+mosquito+transmission+success+in+the+rodent+malaria+parasite+Plasmodium+chabaudi+adami.">A study on pathogenicity and mosquito transmission success in the rodent malaria parasite Plasmodium chabaudi adami.</a> Int J Parasitol. 39, 347-354 (2009).</li><li>Pattaradilokrat, S., Culleton, R. L., Cheesman, S. J., Carter, R. G. e. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=encoding+erythrocyte+binding+ligand+linked+to+blood+stage+multiplication+rate+phenotype+in+Plasmodium+yoelii+yoelii.">encoding erythrocyte binding ligand linked to blood stage multiplication rate phenotype in Plasmodium yoelii yoelii.</a> Proc Natl Acad Sci U S A. 106, 7161-7166 (2009).</li><li>Pattaradilokrat, S., Cheesman, S. J., Carter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linkage+group+selection:+towards+identifying+genes+controlling+strain+specific+protective+immunity+in+malaria.">Linkage group selection: towards identifying genes controlling strain specific protective immunity in malaria.</a> PLoS One. 2, e857-e857 (2007).</li><li>Mackinnon, M. J., Read, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Relationships+between+Parasite+Virulence+and+Transmission+in+the+Rodent+Malaria+Plasmodium+chabaudi.">Genetic Relationships between Parasite Virulence and Transmission in the Rodent Malaria Plasmodium chabaudi.</a> Evolution. 53, 689-703 (1999).</li></ol>

כי המחברים הם עובדי ממשלה וזה עובד ממשלה, העבודה היא בתחום הציבורי בארצות הברית. על אף כל הסכם אחר, NIH שומרת לעצמה את הזכות למסור את העבודה PubMedCentral לתצוגה ושימוש על ידי הציבור, עשויה PubMedCentral תג או לשנות את שיטות העבודה עולה בקנה אחד עם המקובל. אתה יכול להקים זכויות מחוץ לארה&quot;ב בנושא רישיון להשתמש הממשלה.

אנו מדגימים את הטכניקות לייצור צלב גנטי המלריה Plasmodium מכרסם yoelii, שגם הוא החלים על ייצור של צלבים גנטי malarias מכרסמים אחרים. זיהומים של עכברים עם שיבוטים אחד ההורים מבוצעים בדרך כלל כדי לקבוע שידור מוצלח של טפילים ההורים על מנת להבטיח כי ההורים הם המוסמכים בייצ?...

משלים חומרים: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תחזוקה של עכברי מעבדה </li><li style=";text-align:right;direction:rtl"> תחזוקה של המעבדה יתושים </li><li style=";text-align:right;direction:rtl"> בבדיקה מיקרוסקופית של כתמי דם דק מוכתמים Giemsa כתם </li><li style=";text-align:right;direction:rtl"> מדידת צפיפות תא דם אדום </li></ul> תחזוקה של עכברי מעבדה נקבות של זן עכבר מעבדה מולדת BALB / c, בגילאי 5 עד 8 שבועות הישן, הם השתמשו במחקר. עכברים הם שוכנו כלוב סטנדרטי פוליקרבונט מוצק תחתית עם מכסה מתכת בר, מצויד במזין ובקבוק מים. עכברים נשמרים בטמפרטורה קבועה (25 ± 1 ° C) על שעה 12:12 אור: מחזור כהה. עכברים מותר להאכיל על 2018S הרלן Teklad גלובל 19% חלבון בתזונה extruded מכרסם (sterilizable; הארלן מ-Teklad) וסיפקה עם שתיית מים כרצונך acidified המודעה. ניסויים בבעלי חיים מתבצעים בהתאם להנחיות והתקנות שנקבעו על ידי טיפול בבעלי חיים ועדת השתמש במכון הלאומי לאלרגיה ומחלות זיהומיות תחת פרוטוקול LMVR11E (National Institutes of Health, Bethesda, מרילנד). תחזוקה של המעבדה יתושים יתושים הם מהמושבה מעבדה-bred של אנופלס stephensi. המבוגרים נשמרים בכלובים ניילון המשיך בחדר טמפרטורה ולחות מבוקרת (23-25 ​​מעלות צלזיוס למשך Plasmodium Plasmodium chabaudi yoelii ו, ו 19-21 ° C עבור Plasmodium berghei; 80-95 לחות%; ב 12:12 שעות אור: מחזור כהה). יתושים בוגרים ניזונים עם גלוקוז 10% ו 2.00% para-aminobenzoic חומצה (PABA) פתרון בתוספת מים. כדי להשיג איכות גבוהה מבוגרים, 500 הזחלים גדלים בתנאי צפיפות נמוכה ב L 1 של מים מזוקקים בצלחת 3 1000 ס&quot;מ לפתוח מסופק עם כ 1 מ&quot;ג של סודיום ביקרבונט. לאחר הבקיעה, הזחלים מקבלים tetramin אבקה (PETCO) עד שהם להתפתח בשלב גולם והם הועברו הכלובים יתושים בוגרים עבור המתעוררים. בבדיקה מיקרוסקופית של כתמי דם דק מוכתמים Giemsa כתם מספריים לגזור את השימוש לנקות את קצה (1.0 מ&quot;מ) של הזנב של העכבר נגועים. מקום אחד ירידה (0.5-1.0 μL) של דם זנב לשקופית טיפוס נקי. עכבר יהיה לעצור את הדימום בתוך 1-2 דקות. המקום שקופיות מפזר נקי על גבי טיפת דם, שמירה על אותה משפחה 45 ° זווית להחליק את הדגימה, ומאפשרים לדם לספוג לרוחב כל מפזר. החזק את השקופית הדגימה לדחוף קדימה את השקופית מפזר במהירות בצורה חלקה לייצר כתם דק. תנו את הסרט דם יבש, ואז לטבול את השקופיות מתנול מוחלט. אפשר להחליק לייבוש באוויר פעם נוספת לפני מכסה אותו עם כתם Giemsa (10% Giemsa צבע במים מזוקקים). לאחר דוגרים הסרטים דם דק 10-15 דקות בטמפרטורת החדר, לשטוף היטב את השקופיות עם מי ברז ולתת לו להתייבש באוויר. בדוק את מספר נגועים בתאי דם אדומים (iRBC; ראה איור 3 עבור המורפולוגיה של RBC נגוע) תחת מיקרוסקופ אור עם שמן טבילה בהגדלה 1000x (עם עדשה אובייקטיבי 100x) ולחשב parasitemia (מספר iRBC ל -100 RBC ספרתי). זנים שונים של טפילי מלריה להשתנות בקצב הצמיחה pathogenicity. מעקב אחר שלב parasitaemias הדם יכול להתבצע 24 שעות לאחר זריקות, תלוי במינון של השלב טפילים בדם מלריה. למשל, בעכברים יהיה מיקרוסקופית חיובית 24 שעות כאשר הוא מוזרק עם 10 7 נגוע RBC intraperitoneally או 10 6 RBC נגוע לווריד. מדידת צפיפות תא דם אדום כמו רמות parasitaemias, תא דם אדום (RBC) צפיפות עכברים נגועים משתנה לאורך הקורס של זיהום. צפיפות RBC יש למדוד בתוך 1-2 שעות לפני תחילת הזיהום יחיד ו מעורב לשבט הניסויים השיבוט. קיימות שתי שיטות למדידת צפיפות RBC: ספירה ידנית באמצעות Neubauer hemocytometer ו ספירה אוטומטית באמצעות Cellometer (Nexcelom Bioscience). בשתי השיטות, לסגת 1 μL דם העכבר הזנב באמצעות זכוכית נימי (VWR) ו לדלל ב 10 מ&quot;ל של PBS ומערבבים היטב. כדי להשתמש hemocytometer Neubauer, עומס 20 μL של ההשעיה על hemacytometer. מניחים את hemacytometer על מיקרוסקופ אור עם עדשה 10x אובייקטיבי. Hemacytometer מכיל רשת מחולק 9 ריבועים גדולים, 4 ריבועים גדולים בפינת מחולקים נוספת לתוך 16 ריבועים קטנים. ספור את המספר הכולל של תאים בכל אחת 16 ריבועים קטנים של ארבעה ריבועים בפינה. כדי למנוע הטיה או לספור ספירת התאים חפיפה קו רשת, לספור תא כמו &quot;ב&quot; אם זה חופף את הקווים העליון או ימין &quot;החוצה&quot; אם זה חופף את השורות למטה או שמאלה. אומדן מספר התאים לכל ריבוע אחד קטן ולחלק ידי 0.00625 (נפח של ריבוע אחד קטן הוא 6.25 NL). זה מניב את מספר התאים לכל microliter (&amp;mu, L). מתוך נתונים אלה, לחשב את צפיפות התאים הסופי אדום דם על ידי הכפלת עם 10,000 (גורם לדילול). שוטפים את תלוש כיסוי תא לספור עם מים מזוקקים ו 70% אתנול; אוויר יבש. לחלופין, לטעון 20 μL של ההשעיה על שקופית ספירת Cellometer קאמרית. הכנס את השקופית לתוך תא שקופיות Cellometer (הקורא). הפעל את התוכנה Cellometer, בחר את &quot;תא דם אדום&quot; אפשרות, והזן גורם לדילול של 10,000. הקלט את צפיפות RBC.

protocol for production of a genetic cross of the rodent malaria parasites

וריאציה בתגובה תרופות נגד מלריה ו pathogenicity של טפילי מלריה היא ביולוגיים והחשיבות רפואי. מיפוי הצמדה הוביל זיהוי מוצלח של גנים או לוקוסים הבסיסית תכונות שונות טפילי מלריה של מכרסמים ובני אדם 1-3 4-6. טפיל המלריה Plasmodium yoelii הוא אחד מיני רבים מלריה מבודד מכרסמים אפריקאי פראי הותאם לגדול במעבדות. מין זה מתרבה רבים מן המאפיינים ביולוגית של טפילי מלריה אדם; סמנים גנטיים כגון microsatellite ו מוגבר אורך קטע פולימורפיזם (AFLP) סמנים יש גם פותחו עבור טפיל 7-9. לכן, מחקרים גנטיים טפילי מלריה מכרסם ניתן לבצע כדי להשלים את המחקר על Plasmodium falciparum. הנה, אנחנו מדגימים את הטכניקות לייצור הכלאה גנטית פ yoelii כי היו הראשונים חלוץ ידי בני הזוג. דוד Walliker, ריצ&#39;רד קרטר, ועמיתיו באוניברסיטה של אדינבורו 10. חוצה גנטיים פ yoelii ושאר טפילי מלריה מכרסם נערכים על ידי הדבקה של עכברים מ.א.ס. מאסכאלאס עם inoculum המכיל gametocytes של שני שיבוטים שונים גנטית שונה פנוטיפים של עניין על ידי מתן יתושים להאכיל על עכברים נגועים 4 ימים לאחר ההדבקה. הנוכחות של gametocytes זכר ונקבה בדם העכבר הוא אישר מיקרוסקופית לפני האכלה. בתוך 48 שעות לאחר האכילה, ב midgut של היתוש, gametocytes הפלואידים להתמיין הגמטות, הזכרית והנקבית, להפרות, וליצור זיגוטה דיפלואידי (איור 1). במהלך הפיתוח של הזיגוטה לתוך ookinete, המיוזה נראה להתרחש 11. אם הזיגוטה נגזר באמצעות הפריה הדדית בין הגמטות של שני טפילים שונים גנטית, חילופי גנטי (reassortment כרומוזומליות ו צולבות בין תורות אי - אחות chromatids של זוג כרומוזומים הומולוגיים;. איור 2) עלולה להתרחש, וכתוצאה מכך רקומבינציה של החומר הגנטי בבית הומולוגיים לוקוסים. הזיגוטה עוברת כל שתי חטיבות הגרעיני רצופים, המוביל four גרעינים הפלואידים. Ookinete נוסף מתפתח oocyst. לאחר oocyst מתבגר, אלפי sporozoites (צאצאיו של הצלב) נוצרות ומשוחרר hemoceal יתושים. Sporozoites נקצרים מן בלוטות הרוק ו מוזרק לארח Murine חדש, שבו פיתוח שלב טרום האריתרוציטים ו האריתרוציטים מתרחש. צורות האריתרוציטים הם משובטים ומסווגים לגבי הדמויות להבחין בקווים ההורים לפני הצמדה מיפוי גנטי. זיהומים בקרה של שיבוטים הורים יחידים מבוצעים בצורה זהה הייצור של צלב גנטית.

Watch this Scientific Journal Video about Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites at JoVE.com

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

חוצה גנטי של טפילי מלריה מכרסם מבוצעות על ידי האכלה two טפילים שונים גנטית יתושים. הצאצאים רקומביננטי הם משובטים מהדם לאחר עכבר המאפשר יתושים לנשוך עכברים נגועים. סרט הווידאו הזה מראה איך לייצר צלבי הגנטי של<em&gt; Plasmodium yoelii</em&gt; והוא החלים על טפילי מלריה אחרים מכרסמים.

פרוטוקול עבור הפקה של הצלב גנטית של טפילי מלריה מכרסמים

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

protocol-for-production-genetic-cross-rodent-malaria

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Immunology and Infection

15.1K Views.  National Institutes of Health. חוצה גנטי של טפילי מלריה מכרסם מבוצעות על ידי האכלה two טפילים שונים גנטית יתושים. הצאצאים רקומביננטי הם משובטים מהדם לאחר עכבר המאפשר יתושים לנשוך עכברים נגועים. סרט הווידאו הזה מראה איך לייצר צלבי הגנטי של Plasmodium yoelii והוא החלים על טפילי מלריה אחרים מכרסמ...

Video: Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

בתחום ההדמיה vivo של טפילים טראנסגנטי שושנת יריחו שורה חיים

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

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 ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

ניתוח כמותי של השפעת כל סוג Hemagglutinins ויראלי ו Neuraminidases באמצעות נוגדנים יוניברסל מבחני כתם פשוט חריץ

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

פשוט פרוטוקול Chelex למיצוי DNA מ<em&gt; אנופלס</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

מניפולציה גנטית ב<em&gt; Δku80</em&gt; זנים לאנליזה גנומית תפקודית של<em&gt; Toxoplasma gondii</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Retroviral תמרה של Helper T תאים כגישה גנטית לחקר מנגנוני בקרת הבידול והתפקוד שלהם

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

תא דם אדום פשוט שיטת תמוגה להקמת שורות תאי B Lymphoblastoid

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

ניתוח פנוטימית של מלריה מכרסם טפיל מינית ושלבי דם מיניים ושלבי יתוש

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

הרומן מזין ללא מערכת לייצור המוני של תאים רוצח טבעי מאתר במבחנה

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

כימות של צמיחה תאיים בתוך המקרופאגים הוא מהיר, שיטה אמינה להערכת של התקפה אלימה של טפילים לישמניה

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...