الشكر لأعضاء لمختبر موينز مدخلات مفيدة أثناء كتابة هذا المقال. هونج كونج هو زميل ما بعد الدكتوراه التي تدعمها المعاهد الوطنية للصحة / معاهد الصحة القومية منح # 5R01HD037909 - 08. CBM هو محقق مع معهد هوارد هيوز الطبي.

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	<li>Westerfield, 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> The Zebrafish Book. , (2000).</li><li>Kimmel, C. B., Warga, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+and+developmental+potential+of+cells+in+the+zebrafish+embryo.">Cell lineage and developmental potential of cells in the zebrafish embryo.</a> Trends Genet. 4, 68-74 (1988).</li><li>Kimmel, C. B., Warga, R. M., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+organization+of+the+zebrafish+fate+map.">Origin and organization of the zebrafish fate map.</a> Development. 108, 581-594 (1990).</li><li>Woo, K., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+and+coherence+in+the+fate+map+of+the+zebrafish+nervous+system.">Order and coherence in the fate map of the zebrafish nervous system.</a> Development. 121, 2595-2609 (1995).</li><li>Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primordial+germ-cell+development:+the+zebrafish+perspective.">Primordial germ-cell development: the zebrafish perspective.</a> Nat Rev Genet. 4, 690-700 (2003).</li><li>Yoon, C., Kawakami, K., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+vasa+homologue+RNA+is+localized+to+the+cleavage+planes+of+2-+and+4-cell-stage+embryos+and+is+expressed+in+the+primordial+germ+cells.">Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells.</a> Development. 124, 3157-3165 (1997).</li><li>Koprunner, M., Thisse, C., Thisse, B., Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+zebrafish+nanos-related+gene+is+essential+for+the+development+of+primordial+germ+cells.">A zebrafish nanos-related gene is essential for the development of primordial germ cells.</a> Genes Dev. 15 (21), 2877-2885 (2001).</li><li>Weidinger, G., Wolke, U., Koprunner, M., Klinger, M., Raz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+tissues+and+patterning+events+required+for+distinct+steps+in+early+migration+of+zebrafish+primordial+germ+cells.">Identification of tissues and patterning events required for distinct steps in early migration of zebrafish primordial germ cells.</a> Development. 126 (23), 5295-5307 (1999).</li><li>Weidinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+zebrafish+primordial+germ+cell+migration+by+attraction+towards+an+intermediate+target.">Regulation of zebrafish primordial germ cell migration by attraction towards an intermediate target.</a> Development. 129 (1), 25-36 (2002).</li><li>Ciruna, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+maternal-zygotic+mutant+zebrafish+by+germ-line+replacement.">Production of maternal-zygotic mutant zebrafish by germ-line replacement.</a> Proc Natl Acad Sci U S A. 99, 14919-14924 (2002).</li><li>Waskiewicz, A. J., Rikhof, H. A., Moens, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eliminating+zebrafish+pbx+proteins+reveals+a+hindbrain+ground+state.">Eliminating zebrafish pbx proteins reveals a hindbrain ground state.</a> Dev Cell. 3, 723-733 (2002).</li><li>Weidinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dead+end,+a+novel+vertebrate+germ+plasm+component,+is+required+for+zebrafish+primordial+germ+cell+migration+and+survival.">dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival.</a> Curr Biol. 13, 1429-1434 (2003).</li><li>Blaser, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+from+non-motile+behaviour+to+directed+migration+during+early+PGC+development+in+zebrafish.">Transition from non-motile behaviour to directed migration during early PGC development in zebrafish.</a> J Cell Sci. 118, 4027-4038 (2005).</li></ol>

السهولة التي يمكن استخدامها لانتاج زرع الوهم المستهدفة هي واحدة من القوى العظمى في الزرد كنموذج الفقاريات. وتعديل هذا البروتوكول لا الموصوفة أعلاه يتيح تحليل وظيفة الجين لجينات الأم مع أدوار أساسية في التنمية لاقحي. منذ هذه الأسماك متحولة لا يمكن البقاء على قيد الحياة حتى سن البلوغ ، فمن الضروري لنقل germline متحولة إلى الجنين إلا المضيف من النوع البري ، وخلق &quot;استنساخ germline&quot; من خلايا متحولة. توليد germline الفسيفساء ينطوي زرع الخلايا الجرثومية البدائية من متبرع متحولة إلى الجنين المضيف من النوع البري. خلافا لجميع الأنساب خلية أخرى في الجنين ، يتم تحديد النسب الخلية الجرثومية في وقت مبكر جدا في التنمية من خلال وراثة المحددات الأمهات 5،6. وبالتالي ، فإن التحدي الأول لصنع فسيفساء germline هو تحديد الخلايا الجرثومية البدائية (PGCs) في جنين المانحة. في مراحل لmidblastula PGCs يقيمون على طول 7،8 الهامش ، واختيار الخلايا بحيث تصل 5-10 عشوائية من الهامش من النسب التي تحمل علامات الجنين المانحة النتائج في كثير من الأحيان في نقل PGCs. عندما تحول إلى قطب من الحيوانات الجنين المضيف غير المسماة ، والخلايا الجرثومية البدائية بنشاط من أجل ترحيل التناسلية الافتراضي حيث يمكن التعرف عليهم بشكل لا لبس فيه على 24 ساعة من التنمية من خلال وضعهم المميز ، كبيرة الحجم ، والاحتفاظ صبغ بسبب بطء معدل التكاثري 9-11. وفي الوقت نفسه ، المستمدة من الجهات المانحة غير PGCs ستكتسب مصير يحدده موقعها في المضيف : الدماغ الأمامي في المقام الأول والعينين إذا كانت تنقل إلى القطب الأريمة الحيوانية. حقن الجنين المضيف مع morpholinos أن نهدم الجين مسدود القضاء على نحو فعال germline المضيفة في الخلية بطريقة مستقلة ، بحيث أنه حتى واحدة من الجهات المانحة المستمدة PGC يمكن إعادة الناس الى germline كامل 10،12 (CM ، والمراقبة الشخصية). في الآونة الأخيرة ، وقد وضعت خط المعدلة وراثيا التي تسمح PGCs تصور في الأجنة الحية خلال مراحل الأريمة 13. عندما عبرت الحدود الى وظيفة الأمومة التي متحولة يتم تحديد ، وهذا التحوير ، جنبا إلى جنب مع morpholinos طريق مسدود ، وجعل زرع germline سهلة لأنه يمكن تحديد PGCs واحدة وزرعت في استضافة germline - المنضب.

الكواشف : تم العثور على وصفات لكثير من الكواشف المستخدمة لهذه الإجراءات في كتاب اسماك الزرد ، والذي يتوفر في الموقع كاملا في <a href="http://zfin.org/zf_info/zfbook/zfbk.html">http://zfin.org/zf_info/zfbook/zfbk.html</a> . وتقدم وصفات لإعداد هناك أسماك المياه ، متوسطة الأجنة مع وبدون المضادات الحيوية ، لpronase dextrans ، dechorionating الفلورسنت ، والسليلوز الميثيل.

generating chimeric zebrafish embryos by transplantation

واحدة من أقوى الأدوات المستخدمة لاكتساب المعرفة في العمليات التنموية المعقدة هو تحليل للأجنة خيالية. يتم تعريف كائن خرافي كما يحتوي على خلايا من اكثر من حيوان واحد ؛ الفسيفساء هي نوع واحد من الوهم تمتزج فيه خلايا من اكثر من التركيب الوراثي ، وعادة من النوع البري ومتحولة. في الزرد ، ويمكن بسهولة جعل الوهم زرع خلايا من الجنين المانحة الى الجنين المضيف في المرحلة الجنينية المناسبة. يتم إنشاء خلايا المسمى المانحة عن طريق الحقن من علامة النسب ، مثل صبغة الفلورسنت ، في مرحلة الجنين واحد الخلية. تتم إزالة الخلايا من الأجنة المانحة المسمى المانحة والمضيفة التي أدخلت أجنة غير المسماة باستخدام النفط التي تسيطر عليها ماصة الزجاج التي شنت على مجمع أو مجهر تشريح. يمكن في بعض الحالات تكون الخلايا المانحة التي تستهدف منطقة محددة أو أنسجة النامية الأريمة أو المعيدة مرحلة الجنين المضيف عن طريق اختيار موقع الزرع في الجنين المضيف على أساس خرائط مصير راسخة.

Watch this Scientific Journal Video about Generating Chimeric Zebrafish Embryos by Transplantation at JoVE.com

Generating Chimeric Zebrafish Embryos by Transplantation

دليل خطوة بخطوة لتوليد اجنة استهدفت الزرد خيالية بواسطة زرع في المرحلة الأريمة أو المعيدة.

توليد الأجنة اسماك الزرد همي بواسطة زرع

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

generating-chimeric-zebrafish-embryos-by-transplantation

Nanyang Technological University

Research

JoVE Journal

Biology

21.1K Views.  Fred Hutchinson Cancer Research Center - FHCRC. دليل خطوة بخطوة لتوليد اجنة استهدفت الزرد خيالية بواسطة زرع في المرحلة الأريمة أو المعيدة.

Video: Generating Chimeric Zebrafish Embryos by Transplantation

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 ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Microinjection الجنين اسماك الزرد إلى تحليل وظائف الجينات

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

الأنسجة الجنينية الوهم المستهدفة : اسماك الزرد المعيدة زرع الخلايا

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

ثنائي الفوتون المستندة إلى تنشيط ضوئي في الأجنة الحية اسماك الزرد

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

زرع GFP - معربا عن Blastomeres للتصوير لايف التنمية الشبكية والدماغ في الجنين اسماك الزرد همي

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

Dechorionation الجنين الميداكا وزرع الخلايا لتوليد الوهم

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Glycans التصوير في الأجنة التي وصفها اسماك الزرد الاستقلابية والكيمياء Bioorthogonal انقر

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

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 ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

الأيض تحليل الملف من الأجنة الزرد

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...