الكتاب أشكر كريستا هاس، دوريس ويبر وبريجيت كورتي للحصول على المساعدة الفنية. ويدعم العمل في المختبر Weidinger من المنح المقدمة من جمعية الألمانية للبحوث WE 4223 / 3-1، WE 4223 / 4-1 ودويتشه FÜR غزلشافت Kardiologie عن طريق أوسكار-لاب-Stipendium وكلاوس جورج اوند-Sigrid- Hengstberger-Forschungsstipendium.

<ol>
	<li>Fang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+profiling+of+cardiomyocytes+identifies+an+early+Jak1/Stat3+injury+response+required+for+zebrafish+heart+regeneration.">Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (33), 13416-13421 (2013).</li><li>Tryon, R. C., Johnson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+of+kit+ligand+a+functional+expression+reveals+lineage-specific+competence+to+promote+melanocyte+rescue+in+the+mutant+regenerating+caudal+fin.">Clonal analysis of kit ligand a functional expression reveals lineage-specific competence to promote melanocyte rescue in the mutant regenerating caudal fin.</a> PloS one. 9 (7), e102317 (2014).</li><li>Wehner, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+defines+organizing+centers+that+orchestrate+growth+and+differentiation+of+the+regenerating+zebrafish+caudal+fin.">Wnt/beta-catenin signaling defines organizing centers that orchestrate growth and differentiation of the regenerating zebrafish caudal fin.</a> Cell Rep. 6 (3), 467-481 (2014).</li><li>Huang, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+expression+of+a+myocardium-specific+transgene+in+zebrafish+transgenic+lines.">Conditional expression of a myocardium-specific transgene in zebrafish transgenic lines.</a> Dev Dyn. 233 (4), 1294-1303 (2005).</li><li>Knopf, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dually+inducible+TetON+systems+for+tissue-specific+conditional+gene+expression+in+zebrafish.">Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (46), 19933-19938 (2010).</li><li>Provost, E., Rhee, J., Leach, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+2A+peptides+allow+expression+of+multiple+proteins+from+a+single+ORF+in+transgenic+zebrafish+embryos.">Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos.</a> Genesis. 45 (10), 625-629 (2007).</li><li>Suster, M. L., Abe, G., Schouw, A., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-mediated+BAC+transgenesis+in+zebrafish.">Transposon-mediated BAC transgenesis in zebrafish.</a> Nat Protoc. 6 (12), 1998-2021 (2011).</li><li>Bussmann, J., Schulte-Merker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+BAC+selection+for+tol2-mediated+transgenesis+in+zebrafish.">Rapid BAC selection for tol2-mediated transgenesis in zebrafish.</a> Development. 138 (19), 4327-4332 (2011).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. (27), (2009).</li><li>Kim, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+cleavage+efficiency+of+a+2A+peptide+derived+from+porcine+teschovirus-1+in+human+cell+lines,+zebrafish+and+mice.">High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.</a> PloS one. 6 (4), e18556 (2011).</li><li>Matthews, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+euthanasia+in+zebrafish.">Anesthesia and euthanasia in zebrafish.</a> ILAR journal / National Research Council, Institute of Laboratory Animal Resources. 53 (2), 192-204 (2012).</li><li>Hyde, D. R., Godwin, A. R., Thummel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+of+morpholinos+into+the+regenerating+adult+zebrafish+tail+fin.">In vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin.</a> J Vis Exp. (61), (2012).</li><li>Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+PCR-ready+genomic+DNA+from+zebrafish+tissues.">Method for isolation of PCR-ready genomic DNA from zebrafish tissues.</a> Biotechniques. 43 (5), 610 (2007).</li><li>Thermes, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=I-SceI+meganuclease+mediates+highly+efficient+transgenesis+in+fish.">I-SceI meganuclease mediates highly efficient transgenesis in fish.</a> Mech Dev. 118 (1-2), 91-98 (2002).</li><li>Suster, M. L., Kikuta, H., Urasaki, A., Asakawa, K., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+in+zebrafish+with+the+tol2+transposon+system.">Transgenesis in zebrafish with the tol2 transposon system.</a> Methods Mol Biol. , 56141-56163 (2009).</li><li>Grabher, C., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meganuclease+and+transposon+mediated+transgenesis+in+medaka.">Meganuclease and transposon mediated transgenesis in medaka.</a> Genome Biol. 8, (2007).</li></ol>

الكتاب ليس لديهم ما يكشف.

الزرد الكبار لديه قدرة مذهلة على تجديد العديد من الأعضاء الداخلية والزوائد بنجاح. فهم شامل للالآليات الجزيئية والخلوية تشارك يتطلب تحليل الأنسجة محددة من وظائف الجينات ومسارات الإشارات. لتحقيق هذا، ويوفر النظام تيتون أداة فعالة للتعبير عن الجينات التي تسيطر عليها ...

الزرد هو كائن نموذج الفقاريات راسخة لدراسة العديد من جوانب التنمية، علم وظائف الأعضاء، والمرض، وتجديد. مع اعتماد متزايد من الزرد كنموذج للعمليات البيولوجية بعد الجنينية، والأدوات التجريبية للمحرض، والتلاعب الأنسجة محددة التعبير الجيني أو مسارات الإشارات أصبحت ذات أهمية متزايدة. بشكل خاص، عانت الدراسات إلى الجهاز وتجديد أطرافهم في الزرد الكبار من عدم وجود أدوات للتشريح من المتطلبات المكانية والزمانية لمسارات الإشارات خلال هذه العمليات التجدد. حاليا، وقد استخدمت ثلاثة أنظمة مختلفة لتحقيق المشروط، الأنسجة محددة التعبير الجيني في تجديد أجهزة الزرد الكبار: نظام جنة المساواة العرقية، السلمون المدخن، والتعبير فسيفساء من حرارة صدمة الجينات المحورة محرض باستخدام بوساطة ينقول transgenesis الجسدية، ونظام تيتون 1 -3. تيتون يشير إلى متغير من التتراسيكلين-تابعتوالت نظام تفعيل النسخي، حيث يتم تنشيط التعبير في وجود المضادات الحيوية التتراسيكلين أو مشتق، على سبيل المثال الدوكسيسيكلين. نظام جنة المساواة العرقية، السلمون المدخن، حيث تم حتى الآن استخدامها في الأسماك البالغة، يعتمد على لجنة المساواة العرقية recombinase تسيطر تاموكسيفين (CreERT2)، الذي يقتصر مكانيا من قبل عناصر تنظيمية الأنسجة محددة التعبير. إزالة لجنة المساواة العرقية يحركها من شريط إيقاف تسهل التعبير عن الجينات في المصالح يقودها المروج التي يجب أن تكون نشطة في جميع أنواع الخلايا 1. إنشاء بوساطة ينقول من الجينات المحورة جسدية أعرب mosaically يوفر نظام لمحرض التعبير التحوير في الأنساب الخلية الفردية. حقن الأجنة الزرد مع ينقول Tol2 يحمل الجين من الفائدة تحت السيطرة النسخي من الصدمة الحرارية النتائج المروج في الأفراد خيالية تحمل عادة التحوير فقط في الأنساب خلية منفصلة من جهاز تجديد 2. في حين أن كلا المنظومتين تتيح للالمشروط الأنسجة جمعية مهندسي البترولcific التعبير الجيني، ونظام لجنة المساواة العرقية، السلمون المدخن لا يمكن عكسها، واستراتيجية باستخدام القائم على ينقول وضع العلامات نسيلي يعاني من طبيعتها العشوائية. وبالتالي، نحن وغيرنا لقد تأقلم مؤخرا أنظمة تيتون المعدلة وراثيا لاستخدامها في الزرد، والتي تسهل زمانيا ومكانيا التعبير الجيني للرقابة التي بالإضافة الانضباطي وعكسها 3-5. يتألف نظام تيتون المستخدمة هنا خط سائق المعدلة وراثيا (TetActivator) فيه دوكسي (DOX) المنشط النسخي -inducible (تحسين transactivator التتراسيكلين العكسي، irtTA، باختصار تيتا) هو تحت سيطرة تسلسل الجينوم التنظيمية الأنسجة محددة. ثانيا، فإنه يتطلب خط الرد المعدلة وراثيا (TetResponder) التي تؤوي الجين من الفائدة تحت السيطرة النسخي المشغل التتراسيكلين (عنصر استجابة تيت؛ TetRE) (الشكل 1A). وهكذا، واستخدام مجموعات محددة من TetActivator وTetResponder خطوط يسمح للالمشروط الأنسجة SPECIالتلاعب المرورية في التعبير الجيني. لقد استخدمت مؤخرا نظام تيتون للتحقيق لوظائف الأنسجة محددة من WNT / بيتا catenin الإشارات في الكبار تجديد الذيل الزعنفة الزرد 3. في البروتوكول المذكورة هنا نحن تصف تدفق العمل لانشاء واستخدام نظام تيتون في الزرد، ولا سيما بالنسبة للدراسات تجديد الزعانف. هذا يتضمن تعليمات مفصلة حول كيفية توليد TetActivator وTetResponder خطوط المعدلة وراثيا مستقرة وبروتوكول لتحريض التحوير في الأجنة والزرد الكبار. وعلاوة على ذلك، وصفنا تقنيات للتحقق من الأنسجة محددة التعبير الجيني في تجديد الزعانف، بما في ذلك بروتوكول لإعداد cryosections زعانف الزرد الكبار. بالإضافة إلى ذلك، نحن نناقش اعتبارات لتصميم التحوير TetActivator، واختيار طريقة transgenesis، والكشف عن TetResponder التعبير. وبالتالي، فإن الهدف العام من هذا البروتوكول هو بمثابة مخططلوضع المتابعة نظام تيتون وظيفية في الزرد لتحقيق المشروط الأنسجة محددة التعبير الجيني، والتي يمكن تطبيقها على أي نوع من الأنسجة في المصالح. أنشأنا ناقلات TetActivator السماح لI-SCEI أو الجيل Tol2 بوساطة خطوط TetActivator مستقرة باستخدام قصيرة تسلسل الجينوم التنظيمية (شظايا محسن؛ Weidinger مختبر قاعدة البيانات البلازميد أي 1247؛ الشكل 1B). يحتوي هذا البناء الكاسيت TetActivator تتكون من البديل متحولة M2 من العكسي تيت مجال كاظمة تنصهر مع فيروس الهربس البسيط VP16 transactivation مجال مشتقة 3F [irtTAM2 (3F)]. التعبير عن TetActivator (تيتا) يمكن رصدها بسهولة لأنها مع AmCyan fluorophore من نفس إطار القراءة المفتوح أعرب المشارك؛ الببتيد P2A يتوسط الريباسي الطفر، والتي ينبغي أن يؤدي إلى إنتاج تيتا وAmCyan كما البروتينات منفصلة في نسبة 1: 1 5،6. يحتوي بناء أيضا poylinker 5 &#39;من TetActivator كاسيت لتسهيل إدراج تسلسل الجينوم التنظيمية ذات الاهتمام باستخدام أساليب الاستنساخ التقليدية. بالإضافة إلى ذلك، أنشأنا بناء تتكون من الكاسيت أعلاه وصف TetActivator بالإضافة إلى مجموعة كاسيت كانامايسين (Weidinger قاعدة البيانات البلازميد مختبر أي 1180؛ الشكل 1C)، والتي يمكن معاد في الكروموسوم الاصطناعي البكتيري (BAC) تحتوي على منطقة الجينومية كبيرة ( عادة في كودون بداية الجين الذي التعبير النمط أن يحاكي قبل التحوير). تتوفر كل من يبني من المختبر Weidinger بناء على طلبها.

<table><tbody><tr><td>Breeding boxes</td><td>Aqua Schwarz</td><td>AquaBox 1</td><td></td></tr><tr><td>Compound fluorescent microscope</td><td>e.g., Leica, Zeiss</td><td>varies with the manufacturer</td><td>to image fluorescent tissue sections</td></tr><tr><td>Confocal microscope</td><td>e.g., Leica, Zeiss</td><td>varies with the manufacturer</td><td>to image fluorescent tissue sections</td></tr><tr><td>Cryostat</td><td>e.g., Leica, Thermo-Scientific</td><td>varies with the manufacturer</td><td>for cryosectioning</td></tr><tr><td>4&amp;rsquo;, 6- diamidino-2-phenylinodole (Dapi)</td><td>Sigma-Aldrich</td><td>D9542</td><td>use 1/5,000 in PBS&lt;br/&gt; for visualization of nuclei</td></tr><tr><td>Doxycycline</td><td>Sigma-Aldrich</td><td>D9891</td><td>prepare stocks in 50% EtOH at 50 mg/ml (97 mM)&lt;br/&gt; for TetResponder induction</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>P6148</td><td>4% (w/v) paraformaldehyde in PBS, pH 7.5&lt;br/&gt; for fixation</td></tr><tr><td>1x Phosphat-buffer saline (PBS)</td><td></td><td></td><td>1.7 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 5.2 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 150 mM NaCl, pH 7.5</td></tr><tr><td>1x Phosphat-buffer saline + Tween 20 (PBT)</td><td></td><td></td><td>1x PBS with 0.1% Tween 20</td></tr><tr><td>Superfrost Ultra Plus adhesion microscope slides</td><td>Thermo Scientific&amp;nbsp;</td><td>1014356190</td><td>for collection of tissue sections</td></tr><tr><td>Stereo fluorescent microscope</td><td>e.g., Leica, Zeiss</td><td>varies with the manufacturer</td><td>for fluorescence-based genotyping</td></tr><tr><td>Thermocycler</td><td>e.g., Biorad, Applied Biosystems</td><td>varies with the manufacturer</td><td>for PCR-based genotyping</td></tr><tr><td>Tissue freezing medium (TFM)</td><td>Triangel Biomedical Sciences</td><td>TFM-C</td><td>for embedding of tissue samples</td></tr><tr><td>Tricaine (L-Ethyl-m-amino-benzoate-methane sulfonate/MS-222)</td><td>Sigma-Aldrich</td><td>E10521</td><td>for anesthesia&lt;br/&gt; use at 1 mg/ml in E3 embryo medium</td></tr></tbody></table>

use of the teton system to study molecular mechanisms of zebrafish regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

لإنشاء نظام تيتون وظيفية لالأنسجة محددة التعبير الجيني محرض، TetActivator المعدلة وراثيا وخطوط TetResponder تحتاج إلى أن تتولد (الشكل 1A). يتم ذلك عن طريق microinjecting TetActivator (الشكل 1B - C) أو TetResponder (الشكل 1E) يبني في الأجنة الزرد المبكرة واللاحقة التك...

Watch this Scientific Journal Video about Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration at JoVE.com

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

استخدام نظام تيتون لدراسة الآليات الجزيئية لتجديد الزرد

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

use-teton-system-to-study-molecular-mechanisms-zebrafish

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

10.6K Views.  Ulm University. Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

Video: Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

التجديد الكلى في اسماك الزرد الكبار قبل جنتاميسين المستحثة الإصابات

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

التقاط إصلاح الأنسجة في الزرد اليرقات مع الوقت الفاصل بين Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

SiRNA نانوحبيبات بوساطة إسكات الجينات في قلب الزرد الكبار

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Transection الحبل الشوكي في اليرقات الزرد

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Neuroscience

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

طعنة الإصابة بالجروح من اسماك الزرد الكبار الدماغ الانتهائي: طريقة المعنية بالتحقيق في تكوين الخلايا العصبية والدماغ الفقارية التجديد

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

فحص تجديد العضلات في نماذج حمار وحشي من أمراض العضلات

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

نموذج إصابة جرح الطعنة للتكتوم البصري البالغ باستخدام الزرد و Medaka للتحليل المقارن للقدرة التجديدية

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

<meta charset="utf8"></head><body>زرع خلايا الزرد القوية لأبحاث التجديد والتطوير

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>تسليط الضوء على المؤلف: رسم خرائط الاتصال الخلوي في الجهاز العصبي لسمك الزرد أثناء التطوير والتجديد

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

زرع الخلايا عالية الدقة في الزرد الجنيني واليرقات

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...

استخدام نظام تيتون لدراسة الآليات الجزيئية لتجديد الزرد

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التجديد الكلى في اسماك الزرد الكبار قبل جنتاميسين المستحثة الإصابات

التقاط إصلاح الأنسجة في الزرد اليرقات مع الوقت الفاصل بين Brightfield Stereomicroscopy

SiRNA نانوحبيبات بوساطة إسكات الجينات في قلب الزرد الكبار

Transection الحبل الشوكي في اليرقات الزرد

طعنة الإصابة بالجروح من اسماك الزرد الكبار الدماغ الانتهائي: طريقة المعنية بالتحقيق في تكوين الخلايا العصبية والدماغ الفقارية التجديد

فحص تجديد العضلات في نماذج حمار وحشي من أمراض العضلات

نموذج إصابة جرح الطعنة للتكتوم البصري البالغ باستخدام الزرد و Medaka للتحليل المقارن للقدرة التجديدية

زرع الخلايا عالية الدقة في الزرد الجنيني واليرقات

زرع الخلايا عالية الدقة في الزرد الجنيني واليرقات

زرع الخلايا عالية الدقة في الزرد الجنيني واليرقات