Dieses Verfahren folgt weitgehend der Transplantation Technik zur Höhle Fische durch das Labor von Bill Jeffrey entwickelt.

Teil 1: Vorbereitung der Embryonen In diesem Protokoll werden wir die jj xy Symbol, um eine hypothetische Zebrafisch-Mutante Linse bezeichnen. <ol><li> Zebrafisch-Stämme sind in Standard-Fisch-Anlage Bedingungen bei 28,5 C auf einem 14h light/10h Dunkel-Zyklus gehalten. </li><li> Am Abend statt Männchen und Weibchen der Zebrafisch-Stamm jj xy: AB / TU; tg (mGFP) in einem Tank mit einem Teiler, um sie voneinander zu trennen. Die Nachkommen dieser Kreuzung wird zum Ausdruck der GFP-Transgen in allen Geweben und wird als Spender verwendet werden. Parallel dazu verwenden die gleiche Prozedur, um Kreuzungen zwischen nicht-transgenen Wildtyp-Tieren. Je nach Zweck dieses Experiments kann man Tiere Wildtyp-Kreuz am jj xy locus als Spender oder Hosts. </li><li> Innerhalb einer Stunde nach dem Licht am Morgen dreht, entfernen Sie die Teiler, um die Fische zu paaren können. </li><li> Nach 15-30 Minuten sammeln Embryonen in 100 x 15 mm Petrischalen, reinigen Sie sie, und ändern Sie das Medium (Ei Wasser). </li><li> Halten Sie die Embryonen in einem 28,5 C Inkubator bis die gewünschte Zeit. </li><li> Wenn Sie bereit sind für eine Transplantation, entfernen Sie das Chorion manuell mit zwei Paaren von scharfen Zangen, und inkubieren die Embryonen in 0,2% EDTA in Calcium-freier ZFR für 30 Minuten. </li></ol> Teil 2: Herstellung von dissection Nadeln <ol><li> Die Transplantation Verfahren erfordert zwei Arten von Nadeln: eine geschärfte eine, die verwendet werden, um Gewebe um die Linse geschnitten, entfernen Sie das Chorion, und lassen Embryonen aus Agarose ist, und einer stumpfen Nadel verwendet werden, um den Spender Linse bewegen und setzen Sie es in den Host. Hier zeigen wir Ihnen, wie Sie diese Nadeln zu machen. </li><li> Zuerst muss die Spitze einer Pasteurpipette abgeschnitten werden mit einer Diamant-Messer. Dann legen Sie eine Glaskapillare in den Pasteur Pipette so dass etwa die Hälfte seiner Länge in ihm ist. Dadurch wird es einfacher, die Wolfram-Draht durch Schmelzen in das Glas in den nächsten Schritt zu immobilisieren. </li><li> Dann führen Sie einen dünnen Wolframdraht in das offene Ende der Kapillare. Melt das Glas über den Draht mit einem Bunsenbrenner. Mit einer Pinzette, stabil halten das Glas Ende mit dem Metalldraht, während Drehen Sie das andere Ende der Nadel mit der Hand. Der erweichte Glas sollte spiralförmig um das Metall Greifen Sie ihn. </li><li> Wenn Sie eine stumpfe Spitze Nadel sind, dann ist dies das Ende des Verfahrens. So erstellen Sie einen geschärften Nadel, halten Sie die Wolfram-Draht über einem Bunsenbrenner für 1-1.5 Minuten, Abbrennen der Metall so eine sehr feine Spitze entsteht. Prüfen Sie die Qualität der Spitze von microscropy.It ist eine gute Idee, beide Nadeln in die Flamme ein paar Sekunden sofort sterilisieren vor der Transplantation. </li></ol> Teil 3: Vorbereitung der Reagenzien <ol><li> Calcium-freie Zebrafisch Ringer (ZFR) Lösungen (116 mM NaCl, 2,9 mM KCl, 5 mM HEPES, pH 7,2) </li><li> 0,2% EDTA in Calcium-freier Zebrafisch Ringer-Lösungen (pH 7,2) </li><li> 1,2% Agarose (niedrige Schmelzpunkt) in Calcium-freier ZFR </li><li> 0,2% Agarose (niedrige Schmelzpunkt) in Calcium-freier ZFR </li><li> Embryo Medium (pH 7,0, pro Liter enthält: 10 ml Hanks Solution # 1, 1 ml Hanks Solution # 2, 10ml Hanks Solution # 4, 10ml Hanks Solution # 5, 0,35 g Natriumbicarbonat, 300 uL von 2M HCl, Penicillin-Streptomycin 500.000 U) wie in der Zebrafisch-Book (University of Oregon Press, 2000). </li><li> Tungsten Draht, vergoldet, 0,1 mm Durchmesser, Alfa Aesar </li><li> Kapillarrohr, 1,0 mm Durchmesser, World Precision Instruments </li></ol> Teil 4: Transplantation Verfahren <ol><li> In einer Kunststoff-Zentrifugenröhrchen, bereiten 1,2% Agarose für niedrigen Schmelzpunkt in Calcium-freier ZFR vorgewärmt auf 40 C in einem Wasserbad. </li><li> Bei der gewünschten Stufe (30 hpf, zum Beispiel), nehmen die Embryonen in einer Pasteurpipette langsam zu vertreiben sie in die Zentrifugenröhrchen, und langsam zu vertreiben sie in die Zentrifugenröhrchen und Inkubation für ca. 5 Sekunden. Die Spender und Techniker sollten getrennt inkubiert werden. </li><li> Mit einer Pipette nehmen die Embryonen in 1,2% Agarose aus dem Zentrifugenröhrchen und ordnen sie in zwei parallelen Reihen in einem 100mm Polystyrol-Petrischale, halten Wirte und Spender zu trennen. Durch Pipettieren sie langsam und sorgfältig, werden die meisten Embryonen auf ihren Seiten in der Agarose liegen. Sie müssen diejenigen, die nicht in dieser Position liegen, mit der stumpfen Nadel so, dass das Auge nach oben, bevor die Agarose erstarrt neu zu orientieren. </li><li> Warten Sie auf die 1,2% Agarose zu verfestigen, und dann überlagern sie mit 0,2% niedrig schmelzenden Agarose-Lösung. </li><li> Mit dem geschärften Nadel, entfernen Sie alle Agarose um die Augen, und sehr vorsichtig schneiden Sie die Linse aus dem Host und Spender Embryonen mit kleinen Strichen. Achten Sie darauf, nur nahe genug geschnitten, um das Objektiv, es nicht zu zerreißen, aber auch so nicht zu entfernen zuviel Gewebe aus dem Rest der Host Auge. </li><li> Einmal frei, wird Linsen in das Medium schweben. Mit der stumpfen Nadel vorsichtig drücken Sie den SpenderObjektiv bis knapp über den Ort, an dem der Host-Objektiv würde normalerweise, und dann drücken Sie ihn in das Auge mit der stumpfen Nadel. Der Host-Objektiv kann verworfen werden. </li><li> Lassen Spender und Wirt Embryonen in 1,2% Agarose für 30-60 Minuten, dann lassen Sie sie von der Agarose mit der spitzen Nadel, und übertragen Sie sie in eine 24-Well-Platte mit Embryo Medium. Jeder Embryo sollte einen getrennten gut. Achten Sie darauf, in die Vertiefungen richtig, so dass es klar ist, welche Host, mit dem Spender entspricht Etikett. </li><li> Inkubieren bei 28,5 C. Der Spender stammenden Linse aus dem Host-Objektiv kann auf der nicht operierten Seite des gleichen Tieres auf GFP-Fluoreszenz unterschieden werden. Die Abschnitte können auch auf die Linse Größe zu messen und festzustellen, ob es richtig differenziert werden. </li></ol><img src="/files/ftp_upload/1258/1258fig1.jpg" alt="Abbildung 1" /> Abb.. 1 Low Vergrößerung Bild eines geschärften Nadel für Objektiv Transplantation verwendet. Eine Glaskapillare (B) wird in die Pasteurpipette (A) eingesetzt und ein dünner Wolframdraht (C) ist in das offene Ende der Kapillare eingesetzt. Das Glas über den Draht wird mit einem Bunsenbrenner aufgeweicht. Mit einer Pinzette, stabil halten das Glas Ende mit dem Metalldraht, während Drehen Sie das andere Ende des Pasteur-Pipette mit der Hand. Der erweichte Glas sollte spiralförmig um die Metall-, Greifen Sie ihn. <img src="/files/ftp_upload/1258/fig2 1258.png" alt="" width="600" height="204" /> Abb.. 2 Die Spitzen der beiden Arten von Nadeln für Objektiv Transplantation verwendet. <img src="/files/ftp_upload/1258/1258fig3.jpg" alt="Abbildung 3" /> Abb.. 3 Embryonen unterzog Objektiv Transplantation bei 30hpf. Gezeigt sind ein Spender Embryo (A, B), eine Vielzahl Auge mit transplantierten Linse (C, D), und der Seite der Steuerung des Host-Embryo (E, F). Jedes Auge ist in beiden Durchlicht und UV-Beleuchtung gezeigt, wie angegeben. Die Fotografien wurden bei 48 hpf genommen. Das Q01 [9] transgene Linie wurde in diesem Experiment verwendet.

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

Mehrere Schritte erfordern besondere Aufmerksamkeit bei der Linse Transplantationen. <ol><li> Geschärft Nadeln: Es ist besser, mehrere Nadeln vor der Durchführung Objektiv Transplantation vorzubereiten, und die Spitze der Nadeln sind so dünn wie möglich. Eine dickere Nadel reißt mehr Gewebe wegen seiner größeren Durchmesser, so dass ein Ausfall in die Augen zu heilen. </li><li> Die Anordnung der Embryonen: Kurz vor der Transplantation müssen die Embryonen Position, so dass sie auf ihren Seiten liegend sind. ...

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<td>0.1mm diameter tungsten wire</td>
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lens transplantation in zebrafish and its application in the analysis of eye mutants

Das Objektiv spielt eine wichtige Rolle in der Entwicklung der Augenbecher [1,2]. Mit der Zebrafisch als Modellorganismus, können Fragen zur Linse Entwicklung angesprochen werden. Der Zebrafisch ist nützlich für genetische Studien durch mehrere vorteilhafte Eigenschaften, einschließlich der kleinen Größe, hohe Fruchtbarkeit, kurzen Lebenszyklus und Pflegeleichtigkeit. Objektiv Entwicklung erfolgt rasch im Zebrafisch. Um 72 HPF, ist die Zebrafisch-Objektiv funktionell ausgereift [3]. Reichlich genetischen und molekularen Ressourcen zur Verfügung stehen, die Forschung im Zebrafisch zu unterstützen. Darüber hinaus bietet die Ähnlichkeit der Zebrafisch-Auge mit denen anderer Wirbeltiere Grundlage für seine Verwendung als ein hervorragendes Tiermodell der menschlichen Mängel [4-7]. Mehrere Zebrafisch-Mutanten weisen Objektiv Anomalien, einschließlich hohe Zelltod, der in einigen Fällen zu einer vollständigen Degeneration der Linse Gewebe [8]. Um festzustellen, ob Objektiv Anomalien aufgrund von inneren Ursachen oder fehlerhafter Interaktionen mit dem umgebenden Gewebe, ist die Transplantation einer Mutante Objektiv in einem Wildtyp-Auge durchgeführt. Mit Feuer-poliertem Metall Nadeln, mutierte oder Wildtyp-Objektive sind sorgfältig aus dem Spendertier seziert, und in die Host. Zur Unterscheidung Wildtyp und Mutante Gewebe, ist eine transgene Linie als Spender verwendet. Diese Linie drückt Membran-gebundenen GFP in allen Geweben, einschließlich der Linse. Diese Transplantation Technik ist ein wichtiges Instrument in den Studien von Zebrafisch-Mutanten-Objektiv.

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

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

Objektiv Entwicklung beinhaltet Wechselwirkungen mit anderen Geweben. Mehrere Zebrafisch Auge Mutanten sind durch eine abnormal kleine Linse Größe aus. Hier zeigen wir eine Linse Transplantation experimentieren, um festzustellen, ob dieser Phänotyp durch inneren Ursachen oder defekte Interaktion mit Gewebe, die das Objektiv umgeben ist.

Objektiv Transplantation in Zebrafisch und ihre Anwendung in der Analyse von Eye-Mutanten

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

lens-transplantation-zebrafish-its-application-analysis-eye

Research

JoVE Journal

Biology

11.0K Aufrufe.  The Second Teaching Hospital of Jilin University. Objektiv Entwicklung beinhaltet Wechselwirkungen mit anderen Geweben. Mehrere Zebrafisch Auge Mutanten sind durch eine abnormal kleine Linse Größe aus. Hier zeigen wir eine Linse Transplantation experimentieren, um festzustellen, ob dieser Phänotyp durch inneren Ursachen oder defekte Interaktion mit Gewebe, die das Objektiv umgeben ist.

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

Transplantation of Whole Kidney Marrow in Adult Zebrafish

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

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

Die Transplantation von Whole Kidney Marrow in Adult Zebrafisch

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

Forschung

Biologie

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

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

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

Vorbereitung und Kultur der Rat Linsenepithelzellen Explantate für ein Studium der terminalen Differenzierung

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

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

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

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

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

Primäre Kultur und Plasmid Elektroporation der Murine Corti-Organ.

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

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

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

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

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

In-vivo-Elektroporation von Morpholinos in die Adult Zebrafisch Retina

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 Adult Zebrafisch

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

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

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

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

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

Design und Analyse des Temperaturpräferenzverhaltens und seines circadianen Rhythmus in Drosophila

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

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

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

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

Analyse der Nephron Zusammensetzung und Funktion im erwachsenen Zebrafisch Nieren

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

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

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

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

Sequential Anwendung von Glasplättchen die Drucksteifigkeit der Maus Objektiv zu beurteilen: Stamm und morphometrische Analysen

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

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

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

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

Hochauflösende Zeitraffer-Imaging und automatische Analyse von Dynamik der Mikrotubuli in lebenden menschlichen Endothelzellen aus der Nabelvene

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

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

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

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

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

Die Montage und Anwendung der &quot;Shear Ringe &#39;: A Novel Endothelial Modell für Orbital, unidirektional und periodische Strömungs- und Scherspannung

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