הליך זה כדלקמן במידה רבה את טכניקת השתלת שפותחה עבור דג המערה על ידי המעבדה של ביל ג&#39;פרי.

חלק 1: הכנת עוברים בפרוטוקול זה, נשתמש בסמל JJ xy לציון מוטציה היפותטי העדשה דג הזברה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זני דג הזברה נשמרים בתנאים מתקן סטנדרטי דגים ב-C 28.5 על מחזור light/10h 14h כהה. </li><li style=";text-align:right;direction:rtl"> בערב, זכרים ונקבות מקום של ג&#39;יי ג&#39;יי זן דג הזברה xy: AB / TU; TG (mGFP) במיכל עם מחיצה כדי להפריד אותם אחד מהשני. הצאצאים של צלב זה יבטא את transgene GFP ברקמות כולם, ישמש תורמים. במקביל, השתמש בפרוצדורה זהה כדי להגדיר חוצה בין הלא מהונדס wild-type בעלי חיים. בהתאם לצורך ניסוי זה, אפשר לחצות את חיות הבר מסוג ב מוקד JJ xy כמו תורמים או המארחים. </li><li style=";text-align:right;direction:rtl"> תוך שעה אחת לאחר שהאור נדלק בבוקר, להסיר את המחיצות כדי לאפשר דגים להזדווג. </li><li style=";text-align:right;direction:rtl"> לאחר 15-30 דקות לאסוף עוברים 100 x 15 מ&quot;מ צלחות פטרי, לנקות אותם, ולשנות את המדיום (מים ביצה). </li><li style=";text-align:right;direction:rtl"> שמור את העוברים 28.5 C החממה עד הזמן הרצוי. </li><li style=";text-align:right;direction:rtl"> כאשר מוכן להשתלה, להסיר את סיסית ידני עם שני זוגות מלקחיים חדים, דגירה עוברים EDTA 0.2% ב-ZFR סידן חינם במשך 30 דקות. </li></ol> חלק 2: הכנת מחטים לנתיחה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הליך ההשתלה דורשת שני סוגים של מחטים: אחת חידד, המשמש לחתוך הרקמות הסובבות את העדשה, להסיר את סיסית, ועוברים שחרור agarose וכן מחט קהה המשמש כדי להזיז את העדשה התורם להכניס אותו לתוך המחשב המארח. כאן אנו יראה לכם כיצד לעשות את המחטים. </li><li style=";text-align:right;direction:rtl"> ראשית, קצה פיפטה פסטר חייבים להיות מנותקים באמצעות סכין יהלום. ואז להוסיף כוס נימי אל קצה פיפטה פסטר כך כמחצית אורכו בתוכו. זה יקל לשתק את החוט טונגסטן על ידי המסת אותו לתוך הכוס בשלב הבא. </li><li style=";text-align:right;direction:rtl"> ואז להוסיף תיל טונגסטן דק לתוך סוף הפתוחים של נימי. ממיסים את הכוס על חוט עם מבער בונזן. בעזרת מלקחיים, להחזיק בסוף יציב את הכוס עם חוט המתכת תוך פיתול את הקצה השני של המחט עם היד שלך. הזכוכית מרוככת ספירלה סביב המתכת צריך מרתק אותו במקום. </li><li style=";text-align:right;direction:rtl"> אם אתה עושה מחט קצה קהה, אז זה הסוף של ההליך. כדי ליצור מחט חידד, החזק את החוט טונגסטן מעל מבער בונזן עבור 1-1.5 דקות, שורף את המתכת כך טיפ יפה מאוד נוצר. בדוק את איכות טיפ על ידי microscropy.It הוא רעיון טוב לעקר הן מחטים הלהבה לכמה שניות מיד לפני ההשתלה. </li></ol> חלק 3: הכנת ריאגנטים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> סידן חינם (ZFR) דג הזברה Ringer של פתרונות (116 mM NaCl, KCl mM 2.9, 5 HEPES מ&quot;מ, pH 7.2) </li><li style=";text-align:right;direction:rtl"> 0.2% EDTA בסידן ללא פתרונות דג הזברה של רינגר (pH 7.2) </li><li style=";text-align:right;direction:rtl"> 1.2% agarose (נקודת התכה נמוכה) ב-ZFR סידן חינם </li><li style=";text-align:right;direction:rtl"> 0.2% agarose (נקודת התכה נמוכה) ב-ZFR סידן חינם </li><li style=";text-align:right;direction:rtl"> בינוני עובריים (pH 7.0, לליטר מכיל: 10 מ&quot;ל פתרון הנקס # 1, 1ml פתרון הנקס # 2, פתרון 10ml הנקס # 4, הפתרון 10ml הנקס # 5, 0.35 גרם נתרן ביקרבונט, 300 UL של 2M HCl, פניצילין, סטרפטומיצין 500.000 U) כפי שמתואר בספר דג הזברה (אוניברסיטת אורגון Press, 2000). </li><li style=";text-align:right;direction:rtl"> תיל טונגסטן, מצופה זהב, בקוטר של 0.1mm, אלפא Aesar </li><li style=";text-align:right;direction:rtl"> צינור נימי, בקוטר של 1.0mm, Precision Instruments העולם </li></ol> חלק 4: הליך השתלה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בתוך צינור פלסטיק צנטריפוגות, להכין 1.2% נמוך נקודת התכה agarose ב-ZFR סידן חופשי שחומם מראש ל-C 40 באמבט מים. </li><li style=";text-align:right;direction:rtl"> בשלב הרצוי (30 hpf, למשל), לקחת את העוברים פיפטה פסטר, לאט לגרש אותם לתוך הצינור צנטריפוגות, ולאט לאט לגרש אותם לתוך הצינור צנטריפוגות ו דגירה במשך כ 5 שניות. התורמים ומארחים צריך להיות מודגרות בנפרד. </li><li style=";text-align:right;direction:rtl"> עם טפטפת, לקחת את העוברים agarose 1.2% מהצינור צנטריפוגות ומסדרים אותם בשתי שורות מקבילות צלחת פטרי 100mm קלקר, שמירה המארחים ותורמים נפרד. עד pipetting אותם לאט ובזהירות, רוב העוברים ישקר על צדם של agarose. יהיה עליך לכוון את אלה שאינם שוכבים בתנוחה זו באמצעות מחט קהה כך העין כלפי מעלה לפני agarose מתמצק. </li><li style=";text-align:right;direction:rtl"> המתן agarose 1.2% לחזק ולאחר מכן כיסוי זה עם 0.2% נקודת התכה נמוכה פתרון agarose. </li><li style=";text-align:right;direction:rtl"> באמצעות מחט חידד, להסיר כל agarose סביב העיניים, מאוד בזהירות לחתוך את העדשה מן המארח עוברים התורם באמצעות משיכות קטנות. הקפד לחתוך מספיק קרובים העדשה כדי לא לקרוע אותו, אבל גם אז אתה לא להסיר רקמה מדי משאר עין המארח. </li><li style=";text-align:right;direction:rtl"> ברגע חופשי, עדשות יצוף במדיום. באמצעות מחט בוטה, בזהירות לדחוף את התורםהעדשה בדיוק מעל המיקום של איפה לארח את העדשה יהיה כרגיל, ולאחר מכן לדחוף אותו לתוך העין עם המחט קהה. העדשה עשויה לארח להיות מושלך. </li><li style=";text-align:right;direction:rtl"> השאר עוברים התורם המארח agarose 1.2% עבור 30-60 דקות, ואז לשחרר אותם מן agarose באמצעות מחט חדה, ולהעביר אותם לתוך צלחת של 24 באר עם המדיום העובר. כל העובר צריך לכבוש גם נפרדים. הקפד לתייג את בארות כראוי כך ברור אשר מתאים לארח אשר תורם. </li><li style=";text-align:right;direction:rtl"> דגירה ב 28.5 ג התורם הנגזרות העדשה ניתן להבחין בין העדשה מארח בצד unoperated של החיה אותו מבוסס על ה-GFP פלואורסצנטי. מדורים אפשר גם למדוד את גודל העדשה כדי לקבוע אם הוא מבדיל כראוי. </li></ol><img src="/files/ftp_upload/1258/1258fig1.jpg" alt="דמות 1" /> איור. 1 תמונה בהגדלה נמוכה של מחט חידד המשמש השתלת עדשה. זכוכית נימי (ב) מוכנס לתוך פיפטה פסטר (א), וכן חוט דק טונגסטן (C) מוכנס לתוך סוף הפתוחים של נימי. הזכוכית מעל החוט הוא התרכך עם מבער בונזן. בעזרת מלקחיים, להחזיק בסוף יציב את הכוס עם חוט המתכת תוך סובב את הקצה השני של פיפטה פסטר עם היד שלך. הזכוכית מרוככת צריך ספירלה סביב המתכת, לופת אותו במקום. <img src="/files/ftp_upload/1258/fig2 1258.png" alt="" width="600" height="204" /> איור. 2 טיפים של שני סוגים של מחטים המשמשים השתלת עדשה. <img src="/files/ftp_upload/1258/1258fig3.jpg" alt="דמות 3" /> איור. 3 עוברים עברה השתלת עדשה ב 30hpf. המוצגים הם עובר התורם (A, B), עין מארח עם עדשה המושתלים (C, D), ואת הצד מלאה של העובר מארח (E, F). כל עין מוצג באור הן מועבר תאורה UV כמצוין. התמונות צולמו ב hpf 48. Q01 [9] קו מהונדס נעשה שימוש בניסוי הזה.

<ol>
	<li>Thut, C. J., Rountree, R. B., Hwa, M., Kingsley, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+in+situ+screen+provides+molecular+evidence+for+the+induction+of+eye+anterior+segment+structures+by+the+developing+lens.">A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens.</a> Dev Biol. 231, 63-76 (2001).</li><li>Yamamoto, Y., Jeffery, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+role+for+the+lens+in+cave+fish+eye+degeneration.">Central role for the lens in cave fish eye degeneration.</a> Science. 289, 631-633 (2000).</li><li>Easter, S. S., Nicola, 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=The+development+of+vision+in+the+zebrafish(Danio+rerio).">The development of vision in the zebrafish(Danio rerio).</a> Dev Biol. 180, 646-663 (1996).</li><li>Schmitt, E., Dowling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+eye+morphogenesis+in+the+zebrafish.+Brachydanio+rerio.">Early eye morphogenesis in the zebrafish. Brachydanio rerio.</a> J.comp. Neuro. 344 (4), 532-542 (1994).</li><li>Malicki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+power+of+forward+genetics--analysis+of+neuronal+diversity+and+patterning+in+the+zebrafish+retina.">Harnessing the power of forward genetics--analysis of neuronal diversity and patterning in the zebrafish retina.</a> Trends Neurosci. 23 (11), 531-541 (2000).</li><li>Malicki, J., Pujic, Z., Thisse, C., Thisse, B., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+and+reverse+genetic+approaches+to+the+analysis+of+eye+development+in+zebrafish.">Forward and reverse genetic approaches to the analysis of eye development in zebrafish.</a> Vision Res. 42 (4), 527-533 (2002).</li><li>Avanesov, A., Malicki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+study+neurogenesis+in+the+zebrafish+retina.">Approaches to study neurogenesis in the zebrafish retina.</a> Methods Cell Biol. 76, 333-384 (2004).</li><li>Vihtelic, T. S., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+mutagenesis+yields+eye+morphological+mutants+with+retinal+and+lens+defects.">Zebrafish mutagenesis yields eye morphological mutants with retinal and lens defects.</a> Vision Res. 42, 535-540 (2002).</li><li>Godinho, L., Mumm, J. S., Williams, P. R., Schroeter, E. H., Koerber, A., Park, S. W., Leach, S. D., Wong, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+amacrine+cell+neurites+to+appropriate+synaptic+laminae+in+the+developing+zebrafish+retina.">Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina.</a> Development. Nov 132, 5069-5079 (2005).</li></ol>

צעדים אחדים דורשים תשומת לב מיוחדת במהלך השתלות העדשה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חידד מחטים: עדיף להכין מחטים מספר לפני ביצוע השתלת עדשה, וקצה מחטים צריך להיות דק ככל האפשר. מחט עבה יקרע רקמות יותר בגלל קוטר רחב יותר שלה, גורם לכישלון בעין לרפא...

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<tr>
<td>0.1mm diameter tungsten wire</td>
<td>&nbsp;</td>
<td>A Johnson Matthey</td>
<td>45086</td>
<td>&nbsp;</td>
<tr>
<td>Agarose(low melting point)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>39346</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass capillaries</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW 100-4</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Dumont #5</td>
<td>11252-30</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur pipette</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-20C</td>
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<td>Pipette pump</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-683C</td>
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<td>Petri Dish</td>
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<td>Penicillin-streptomycin</td>
<td>&nbsp;</td>
<td>GIBCO</td>
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lens transplantation in zebrafish and its application in the analysis of eye mutants

העדשה משחק תפקיד חשוב בהתפתחות של כוס הראייה [1,2]. השימוש דג הזברה כמודל אורגניזם, שאלות לגבי התפתחות העדשה ניתן לטפל. דג הזברה הוא שימושי עבור מחקרים גנטיים בשל יתרון מאפיינים, ובהם גודל קטן, פוריות גבוהה, מחזור חיים קצר, וקלות לטיפול. פיתוח עדשה מתרחשת במהירות דג הזברה. עד hpf 72, העדשה דג הזברה הוא בוגר תפקודית [3]. משאבים גנטיים מולקולריים שופע זמינים כדי לתמוך במחקר דג הזברה. בנוסף, הדמיון של העין דג הזברה לאלה של חוליות אחרים מספק בסיס השימוש בו במודל חיה מעולה של פגמים אנושיים [4-7]. מוטנטים דג הזברה מספר מפגינים ליקויים העדשה, כולל רמות גבוהות של מוות של תאים, אשר בחלק מהמקרים גורמת לניוון מוחלט של רקמות העדשה [8]. כדי לקבוע אם הפרעות העדשה הן בשל גורם פנימי או פגום אינטראקציות עם הרקמות הסובבות, השתלת עדשה לתוך העין מוטציה wild-type מבוצע. באמצעות אש מלוטש מחטים מתכת, עדשות מוטנטי או wild-type הם גזור בקפידה מן החי התורם, והועבר לתוך המארח. כדי להבדיל בין הרקמות wild-type ו - מוטציה, קו מהונדס משמש התורם. קו זה מבטא קרום הנכנס GFP ברקמות, כולל העדשה. טכניקה זו השתלה היא כלי חיוני במחקרים של מוטנטים דג הזברה העדשה.

Watch this Scientific Journal Video about Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants at JoVE.com

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

העדשה כוללת פיתוח אינטראקציות עם רקמות אחרות. מספר מוטציות עין דג הזברה מתאפיינים בגודל העדשה קטן באופן חריג. כאן אנו מדגימים השתלת עדשה הניסוי כדי לקבוע אם פנוטיפ זה נובע גורם פנימי או אינטראקציות עם רקמות פגומות המקיפים את העדשה.

השתלת עדשה של דג הזברה Application שלה ניתוח של מוטאנטים העין

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

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JoVE Journal

Biology

11.0K Views.  The Second Teaching Hospital of Jilin University. העדשה כוללת פיתוח אינטראקציות עם רקמות אחרות. מספר מוטציות עין דג הזברה מתאפיינים בגודל העדשה קטן באופן חריג. כאן אנו מדגימים השתלת עדשה הניסוי כדי לקבוע אם פנוטיפ זה נובע גורם פנימי או אינטראקציות עם רקמות פגומות המקיפים את העדשה.

Video: Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

השתלה של מח כליות שלמים דג הזברה למבוגרים

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

הכנה והתרבות של עדשה Explants עכברוש אפיתל ללימוד בידול Terminal

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

תרבות ראשיים electroporation פלסמיד של האיבר Murine של קורטי.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

ב electroporation vivo של Morpholinos לתוך הרשתית דג הזברה למבוגרים

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging דג הזברה למבוגר

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

עיצוב וניתוח של התנהגות העדפת טמפרטורה והשעון הביולוגי שלה בדרוסופילה

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

ניתוח של הרכב נפרון ותפקוד בכליות דג הזברה למבוגר

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the lens, leading to an increase in the lens&#39; optical power to focus on nearby objects, a process known as accommodation. Age-related changes in lens stiffness have been linked to presbyopia, a reduction in the lens&#39; ability to accommodate, and, by extension, the need for reading glasses. Even though mouse lenses do not accommodate or develop presbyopia, mouse models can provide an invaluable genetic tool for understanding lens pathologies, and the accelerated aging observed in mice enables the study of age-related changes in the lens. This protocol demonstrates a simple, precise, and cost-effective method for determining mouse lens stiffness, using glass coverslips to apply sequentially increasing compressive loads onto the lens. Representative data confirm that mouse lenses become stiffer with age, like human lenses. This method is highly reproducible and can potentially be scaled up to mechanically test lenses from larger animals.

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

יישום סדרתית של הזכוכית Coverslips להעריך את הדחיסה הנוקשת של עדשות העכבר: ניתוח זנים ואת morphometric

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

זמן לשגות הדמיה ברזולוציה גבוהה וניתוח אוטומטי של Dynamics microtubule In Living האדם טבורי וריד לתאי אנדותל

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

האסיפה והיישום של &#39;טבעות Shear&#39;: מודל האנדותל חדשני Orbital, חד-כיוונית תקופתית נוזלי זרימת מתח שאר

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...