فإن الكتاب أود أن أشكر الحياة Freimann مركز العلوم ومركز البحوث للموظفين الزرد لرعايتهم والحفاظ على الزرد.

<ol>
	<li>Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+heart+of+regeneration+in+zebrafish.">Getting to the heart of regeneration in zebrafish.</a> Semin. Cell Dev. Biol. 18, 36-45 (2007).</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=Light-induced+rod+and+cone+cell+death+and+regeneration+in+the+adult+albino+zebrafish+(Danio+rerio)+retina.">Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina.</a> J. Neurobiol. 44, 289-307 (2000).</li><li>Becker, T., Wullimann, M. F., Becker, C. G., Bernhardt, R. R., Schachner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+regrowth+after+spinal+cord+transection+in+adult+zebrafish.">Axonal regrowth after spinal cord transection in adult zebrafish.</a> J. Comp. Neurol. 377, 577-595 (1997).</li><li>Becker, C. G., Becker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repellent+guidance+of+regenerating+optic+axons+by+chondroitin+sulfate+glycosaminoglycans+in+zebrafish.">Repellent guidance of regenerating optic axons by chondroitin sulfate glycosaminoglycans in zebrafish.</a> J. Neurosci. 22, 842-853 (2002).</li><li>Lopez-Schier, H., Hudspeth, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-step+mechanism+underlies+the+planar+polarization+of+regenerating+sensory+hair+cells.">A two-step mechanism underlies the planar polarization of regenerating sensory hair cells.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 18615-1820 (2006).</li><li>Johnson, S. L., Weston, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-sensitive+mutations+that+cause+stage-specific+defects+in+Zebrafish+fin+regeneration.">Temperature-sensitive mutations that cause stage-specific defects in Zebrafish fin regeneration.</a> Genetics. 141, 1583-1595 (1995).</li><li>Nechiporuk, A., Poss, K. D., Johnson, S. L., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positional+cloning+of+a+temperature-sensitive+mutant+emmental+reveals+a+role+for+sly1+during+cell+proliferation+in+zebrafish+fin+regeneration.">Positional cloning of a temperature-sensitive mutant emmental reveals a role for sly1 during cell proliferation in zebrafish fin regeneration.</a> Dev. Biol. 258, 291-306 (2003).</li><li>Tu, S., 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=Fate+restriction+in+the+growing+and+regenerating+zebrafish+fin.">Fate restriction in the growing and regenerating zebrafish fin.</a> Dev. Cell. 20, 725-732 (2011).</li><li>Geraudie, J., Ferretti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+RA-induced+apoptosis+and+patterning+defects+in+regenerating+fins+and+limbs.">Correlation between RA-induced apoptosis and patterning defects in regenerating fins and limbs.</a> Int. J. Dev. Biol. 41, 529-532 (1997).</li><li>Jazwinska, A., Badakov, R., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activin-betaA+signaling+is+required+for+zebrafish+fin+regeneration.">Activin-betaA signaling is required for zebrafish fin regeneration.</a> Curr. Biol. 17, 1390-1395 (2007).</li><li>Laforest, L., Brown, C. W., Poleo, G., Geraudie, J., Tada, M., Ekker, M., Akimenko, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+sonic+hedgehog,+patched+1+and+bmp2+genes+in+patterning+of+the+zebrafish+dermal+fin+rays.">Involvement of the sonic hedgehog, patched 1 and bmp2 genes in patterning of the zebrafish dermal fin rays.</a> Development. 125, 4175-4178 (1998).</li><li>Lee, Y., Grill, S., Sanchez, A., Murphy-Ryan, M., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf+signaling+instructs+position-dependent+growth+rate+during+zebrafish+fin+regeneration.">Fgf signaling instructs position-dependent growth rate during zebrafish fin regeneration.</a> Development. 132, 5173-5183 (2005).</li><li>Poss, K. D., Shen, J., Nechiporuk, A., McMahon, G., Thisse, B., Thisse, C., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+for+Fgf+signaling+during+zebrafish+fin+regeneration.">Roles for Fgf signaling during zebrafish fin regeneration.</a> Dev. Biol. 222, 347-358 (2000).</li><li>Raya, A., Koth, C. M., Buscher, D., Kawakami, Y., Itoh, T., Raya, R. M., Sternik, G., Tsai, H. J., Rodriguez-Esteban, C., Izpisua-Belmonte, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Notch+signaling+pathway+precedes+heart+regeneration+in+zebrafish.">Activation of Notch signaling pathway precedes heart regeneration in zebrafish.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 11889-11895 (2003).</li><li>Smith, A., Avaron, F., Guay, D., Padhi, B. K., Akimenko, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+BMP+signaling+during+zebrafish+fin+regeneration+disrupts+fin+growth+and+scleroblasts+differentiation+and+function.">Inhibition of BMP signaling during zebrafish fin regeneration disrupts fin growth and scleroblasts differentiation and function.</a> Dev Biol. 299, 438-454 (2006).</li><li>Thummel, R., Li, L., Tanase, C., Sarras, M. P., Godwin, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+expression+pattern+and+function+between+zebrafish+hoxc13+orthologs:+recruitment+of+Hoxc13b+into+an+early+embryonic+role.">Differences in expression pattern and function between zebrafish hoxc13 orthologs: recruitment of Hoxc13b into an early embryonic role.</a> Dev. Biol. 274, 318-333 (2004).</li><li>Coonrod, S. A., Bolling, L. C., Wright, P. W., Visconti, P. E., Herr, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+morpholino+phenocopy+of+the+mouse+mos+mutation.">A morpholino phenocopy of the mouse mos mutation.</a> Genesis. 30, 198-200 (2001).</li><li>Heasman, J., Kofron, M., Wylie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-catenin+signaling+activity+dissected+in+the+early+Xenopus+embryo:+a+novel+antisense+approach.">Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach.</a> Dev. Biol. 222, 124-134 (2000).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+knockdown+in+zebrafish.">Effective targeted gene knockdown in zebrafish.</a> Nat. Genet. 26, 216-2120 (2000).</li><li>Hoptak-Solga, A. D., Nielsen, S., Jain, I., Thummel, R., Hyde, D. R., Iovine, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin43+(GJA1)+is+required+in+the+population+of+dividing+cells+during+fin+regeneration.">Connexin43 (GJA1) is required in the population of dividing cells during fin regeneration.</a> Dev. Biol. 317, 541-548 (2008).</li><li>Thummel, R., Bai, S., Sarras, M. P., Song, P., McDermott, J., Brewer, J., Perry, M., Zhang, X., Hyde, D. R., Godwin, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+zebrafish+fin+regeneration+using+in+vivo+electroporation+of+morpholinos+against+fgfr1+and+msxb.">Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb.</a> Dev. Dyn. 235, 336-346 (2006).</li><li>Yin, V. P., Thomson, J. M., Thummel, R., Hyde, D. R., Hammond, S. M., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf-dependent+depletion+of+microRNA-133+promotes+appendage+regeneration+in+zebrafish.">Fgf-dependent depletion of microRNA-133 promotes appendage regeneration in zebrafish.</a> Genes Dev. 22, 728-733 (2008).</li><li>Tawk, M., Tuil, D., Torrente, Y., Vriz, S., Paulin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+gene+transfer+into+adult+fish:+a+new+tool+to+study+fin+regeneration.">High-efficiency gene transfer into adult fish: a new tool to study fin regeneration.</a> Genesis. 32, 27-31 (2002).</li></ol>

ليس لدينا ما يكشف.

هنا، نحن تصف قوية الخسارة من وظيفة إلى نهج البروتينات ضربة قاضية مشروط من الاهتمام خلال تجديد الزعانف في الزرد الكبار. وقد استخدمت هذه التقنية لدراسة الجينات تقاطع الفجوة، مما يشير إلى المستقبلات، عوامل النسخ، و microRNAs خلال ثمرة زعنفة التجدد 16، 20-22. ونحن نتوقع أن هذه التقنية يمكن أيضا أن تستخدم لدراسة الجينات اللازمة لالتئام الجروح وتكوين مأرمة عن طريق تكييف هذه التقنية. على سبيل المثال، نحن وحقن electroporated 1 morpholino التحكم في المسافة بين الزعانف عظمي في النصف الظهري من الزعنفة قبل بتر (الشكل 7). نحن بتر ثم الزعنفة البعيدة على الفور إلى حقن طائرة. 24 HPA، لاحظنا أن الخلايا المستهدفة morpholino هاجروا بشكل أقصى لتشكيل كلا من ظهارة الجرح ومأرمة (الشكل 8)، مشيرا إلى أن الخلايا خلال هذه المراحل المبكرة من التجدد ويمكن أيضا أن targeted. هذه التقنية لديها عدد قليل من القيود الملحوظة. على سبيل المثال، مضان من العلامة فلوريسئين لا تستمر تثبيت التالية وتجهيز المناعية، مما يجعل من المستحيل لربط النمط الظاهري خاص الخلوية (أي تكاثر الخلايا) مع كمية من morpholino الحالي في خلية. وبالإضافة إلى ذلك، لم نتمكن من تحقيق باستمرار electroporation من البلازميدات في تجديد زعنفة الذيل، على الرغم من أن المجموعة السابقة لم تبلغ عن electroporation ناجح من الحمض النووي في أنسجة فنلندا 23. أخيرا، لاحظنا أن morpholino فعالة فقط لمدة 48 ساعة ~ electroporation آخر القرن 21، الذي يحظر استخدام هذه التقنية في شكلها الحالي لاختبار الجينات المسؤولة عن التفريق بين أنواع الخلايا الجديدة. قد يتم إجراء اختبارات إضافية وتعديل إجراءات التغلب على هذه القيود الحالية. وبالإضافة إلى ذلك، فمن الممكن أنه يمكن استخدام هذه التقنيةلاختبار البروتينات التي تنطوي عليها الهجرة خلية من النسيج الأساسي للمأرمة. على سبيل المثال، نحن وحقن electroporated تحكم morpholino إلى جانبي الزعنفة (كما هو موضح في الشكل 7) قبل بتر. نحن بتر ثم النصف الظهري من الزعنفة البعيدة مباشرة إلى الطائرة الحقن ونحن بتر الزعنفة البطنية أكثر من ذلك بكثير بشكل أقصى. في 24 HPA، كانت الخلايا morpholino إيجابية على الجانب الظهري هاجروا من موقع حقن لالمغطي ظهارة الجرح ومأرمة. ومع ذلك، كان هذا لا ينطبق على الجانب البطني (الشكل 9). هذا يدعم الفكرة القائلة بأن فقط الخلايا التي تكمن وراء طائرة بتر المشاركة في استجابة التجدد. هذه البيانات تشير أيضا إلى أن يمكن اختبار البروتينات افترض أن تكون هناك حاجة للهجرة الخلية باستخدام هذه التقنية.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> CUY21-EDIT أو CUY21-SC ساحة electroporator موجة </td><td> بروتك الدولية </td><td> CUY21EDIT أو CUY21SC </td><td> على حد سواء وحدات العمل لهذا البروتوكول </td></tr><tr><td> 3 مم أقطاب قطر مجداف </td><td> بروتك الدولية </td><td> CUY 650-P3 </td><td></td></tr><tr><td> Morpholino </td><td> GeneTools، ذ م م </td><td></td><td> وينبغي Morpholino مصممة خصيصا لبروتين اهتمامك </td></tr><tr><td> 2-فينوكسي ايثانول </td><td> سيغما </td><td> 77861-1L </td><td> التخدير، مخفف 1:1000 في الماء الأسماك لنظام الداخلي، 1:500 للقتل الرحيم </td></tr><tr><td> الدقيقة injectiعلى مضخة </td><td> الآلات الدقيقة العالم </td><td> PV830 هوائي PicoPump </td><td> ويمكن استخدام العديد من أنظمة مختلفة microinjection </td></tr><tr><td> الدقيقة مناور </td><td> الآلات الدقيقة العالم </td><td> MMJR </td><td> اليد اليمنى (MMJL لالوفاض اليسار) </td></tr><tr><td> الدقيقة حقن الإبر، 1.0 ملم خارج القطر </td><td> الآلات الدقيقة العالم </td><td> 1B100F-4 </td><td> هذه هي البورسليكات الزجاج الشعيرات الدموية، وانسحبت إلى إبرة </td></tr><tr><td> إبرة حامل </td><td> الآلات الدقيقة العالم </td><td> 5430-ALL </td><td> بيكو كيت الخرطوم، للتأكد من أن أقحم طوقا 1.0 مم ماصة </td></tr><tr><td> إبرة مجتذب </td><td> سوتر </td><td> P-97 </td><td> وينبغي الأخرى micropipette / إبرة جر العمل أيضا </td></tr><tr><td> مجهر </td><td> لايكا، نيكون، زايسق </td><td> عدد يختلف باختلاف الشركة المصنعة </td><td> أي stereomicroscope مع 20x البصرية والقدرة على العمل مع micromanipulators </td></tr></tbody></table>

in vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin

يمكن أن أنواع معينة من urodeles والأسماك مكتملة العظام تجديد أنسجتها. وأصبحت نموذجا الزرد المستخدمة على نطاق واسع لدراسة تجديد التلقائي للأنسجة البالغين، مثل القلب 1، شبكية العين 2، 3 الحبل الشوكي، العصب البصري 4، خلايا الشعر الحسية 5، وزعانف 6. الزعنفة الزرد هو ملحق بسيطة نسبيا التي يتم التلاعب بها بسهولة لدراسة مراحل متعددة في تجديد تجدد المقطوع. كلاسيكيا، تميزت زعنفة تجديد من قبل ثلاث مراحل متميزة: التئام الجروح، وتشكيل مأرمة، وثمرة زعنفة. بعد بتر جزء من الزعانف، وظهارة المحيطة انتشارا ويهاجر على الجرح. في 33 درجة مئوية، وهذه العملية تحدث في غضون ست ساعات ما بعد بتر (HPA، الشكل 1B) 6،7. المقبل، والخلايا الكامنة من أنساب مختلفة (مثل العظام والدم والدبقية، الخلايا الليفية) إعادة إدخال دورة الخلية لتشكيل مأرمة التكاثري، ثhile البشرة المغطي تستمر في التكاثر (1D الشكل) 8. ثمرة كما يحدث الخلايا الأقرب إلى مأرمة في الأنساب كل منها إعادة التفريق، لتشكيل أنسجة جديدة (الشكل 1E) 8. تبعا لمستوى لبتر، اكتمال تجديد كامل في الاسبوع لمدة شهر. التعبير عن عدد كبير من العائلات الجينات، بما في ذلك hox، wnt، جمعية جيل المستقبل، MSX، حمض الريتينويك، SHH، الشق، BMP، وactivin بيتا الجينات A، هو ما يصل ينظم خلال مراحل معينة من تجديد زعنفة 9-16. ومع ذلك، فقد كان دور كل من هذه الجينات والبروتينات التي ترميز خلال تجديد الصعب تقدير، ما لم يكن المانع محددة للبروتين موجود 13، متحولة حساسة للحرارة أو وجود حيوان المعدلة وراثيا (إما overexpressing البروتين من النوع البري أو المهيمنة 1 سلبية البروتين) قد ولدت 7،12. نحن develoPED تقنية عكس الوراثية بسرعة وسهولة اختبار وظيفة من أي جين خلال تجديد زعنفة. وتستخدم على نطاق واسع [أليغنوكليوتيد] Morpholino لدراسة فقدان بروتينات معينة أثناء الزرد، القيطم والحمص، والفأر التنمية 17-19. Morpholinos basepair مع تسلسل الحمض النووي الريبي مكملة لاما الربط ما قبل مرنا كتلة أو ترجمة مرنا. ونحن تصف طريقة لإدخال بكفاءة فلوريسئين الموسومة العقاقير morpholinos في زعانف اسماك الزرد تجديد في التعبير ضربة قاضية من البروتين الهدف. وmorpholino والصغرى تحقن كل مأرمة من زعنفة الذيل تجديد الزرد وelectroporated في الخلايا المحيطة بها. فلوريسئين يوفر هذا الاتهام إلى electroporate في morpholino وتصور morpholino في الأنسجة زعنفة. هذا البروتوكول يسمح مشروط ضربة قاضية البروتين لدراسة دور بروتينات معينة أثناء نمو زعانف التجدد. في مناقشة:ن، ونحن تصف كيفية ويمكن تكييف هذا النهج لدراسة دور بروتينات معينة أثناء التئام الجروح أو تشكيل مأرمة، فضلا عن علامة المحتملة للهجرة من خلال تشكيل خلية مأرمة.

Watch this Scientific Journal Video about In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin at JoVE.com

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

وصفنا أسلوبا لضربة قاضية مشروط التعبير عن بروتين الهدف خلال الكبار الزرد تجديد زعنفة. هذا الأسلوب الذي ينطوي الصغيرة عن طريق الحقن وelectroporating morpholinos قليل النوكليوتيد العقاقير إلى الأنسجة الزعانف، والذي يسمح باختبار دور هذا البروتين في مراحل مختلفة من تجديد الزعانف، بما في ذلك التئام الجروح، وتشكيل مأرمة، وثمرة التجدد.

في Electroporation المجراة من Morpholinos في الكبار التجديد زعنفة الذيل الزرد

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

in-vivo-electroporation-morpholinos-into-regenerating-adult-zebrafish

Research

JoVE Journal

Biology

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

16.8K Views.  University of Notre Dame. وصفنا أسلوبا لضربة قاضية مشروط التعبير عن بروتين الهدف خلال الكبار الزرد تجديد زعنفة. هذا الأسلوب الذي ينطوي الصغيرة عن طريق الحقن وelectroporating morpholinos قليل النوكليوتيد العقاقير إلى الأنسجة الزعانف، والذي يسمح باختبار دور هذا البروتين في مراحل مختلفة من تجديد الزع...

Video: In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

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.

زرع نخاع الكلى في الجامعة اسماك الزرد الكبار

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

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

وحيد الخلية في الجسم الحي Electroporation داخل الدماغ سليمة النامية

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

الرجعية المدارية في حقن اسماك الزرد الكبار

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

تشريح هيئات من اسماك الزرد الكبار

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

حقن داخل الصفاق الى اسماك الزرد الكبار

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

التسليم المباشر للMorpholinos MIF في Otocyst اسماك الزرد عن طريق الحقن وElectroporation يؤثر على التنمية الداخلية الأذن

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

زرع الخلايا مباشرة في الكلى من اسماك الزرد الكبار

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

تشريح الكلى اسماك الزرد الكبار

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

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.

في الجسم الحي من Electroporation Morpholinos في شبكية العين اسماك الزرد الكبار

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 اسماك الزرد الكبار

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