Questa procedura segue in larga misura la tecnica di trapianto predisposti per i pesci grotta dal laboratorio di Bill Jeffrey.

Parte 1: Preparazione degli embrioni In questo protocollo, useremo il simbolo jj xy per indicare un ipotetico mutante lente zebrafish. <ol><li> Zebrafish ceppi sono mantenuti in condizioni standard impianto di pesce a 28,5 C su un ciclo di 14h light/10h buio. </li><li> In serata, maschi e femmine posto del ceppo zebrafish jj xy: AB / TU; tg (mGFP) in una vasca con un divisore per separare l&#39;uno dall&#39;altro. La progenie di questa croce che esprimono il transgene GFP in tutti i tessuti, e saranno utilizzati come donatori. In parallelo, utilizzare la stessa procedura per creare incroci tra non transgenici wild-type animali. A seconda dello scopo di questo esperimento, si può attraversare animali wild-type a livello del locus jj xy come donatori o host. </li><li> Entro un&#39;ora dopo la luce si accende al mattino, togliere i divisori per consentire al pesce di accoppiarsi. </li><li> Dopo 15-30 minuti raccogliere embrioni in 100 piatti mm x 15 Petri, pulirli, e cambiare il mezzo (acqua uovo). </li><li> Mantenere gli embrioni in C 28,5 incubatore fino al momento desiderato. </li><li> Quando si è pronti per il trapianto, rimuovere il corion manualmente con due paia di pinze taglienti, e incubare gli embrioni nel 0,2% EDTA di calcio senza ZFR per 30 minuti. </li></ol> Parte 2: Preparazione di aghi di dissezione <ol><li> Le procedure di trapianto richiede due tipi di aghi: uno affilato, che viene utilizzato per tagliare tessuti circostanti la lente, rimuovere il corion, e gli embrioni rilascio di agarosio, e un ago smussato utilizzato per spostare la lente dei donatori e inserirlo nella ospitante. Qui vi mostreremo come fare questi aghi. </li><li> In primo luogo, la punta di una pipetta Pasteur deve essere tagliato con un coltello di diamante. Quindi inserire un capillare di vetro nella punta della pipetta Pasteur in modo che circa la metà della sua lunghezza è al suo interno. Questo renderà più facile per immobilizzare il filo di tungsteno da fusione nel bicchiere nella fase successiva. </li><li> Quindi inserire un sottile filo di tungsteno nella parte aperta del capillare. Fate fondere il vetro sopra il filo con un becco Bunsen. Uso di pinze, tenere ferma la fine di vetro con il filo metallico mentre torcendo l&#39;altra estremità dell&#39;ago con la mano. Il vetro dovrebbe ammorbidito a spirale attorno al metallo è presa in posizione. </li><li> Se si stanno facendo un ago punta smussata, allora questa è la fine della procedura. Per creare un ago appuntito, tenere il filo di tungsteno nel corso di un becco Bunsen per 1-1,5 minuti, bruciando il metallo così una punta molto fine è stato creato. Controllare la qualità della punta da microscropy.It è una buona idea per sterilizzare gli aghi sia nella fiamma per alcuni secondi immediatamente prima del trapianto. </li></ol> Parte 3: Preparazione dei reagenti <ol><li> Calcio senza Zebrafish Ringer (ZFR) Soluzioni (116 mM NaCl, 2.9 mM KCl, 5 HEPES mM, pH 7,2) </li><li> 0,2% EDTA di calcio senza soluzioni Zebrafish Ringer (pH 7,2) </li><li> 1,2% di agarosio (basso punto di fusione) di calcio senza ZFR </li><li> 0,2% di agarosio (basso punto di fusione) di calcio senza ZFR </li><li> Medio embrione (pH 7,0, per ogni litro contiene 10 ml di soluzione Hanks # 1, 1 ml Soluzione Hanks # 2, 10 ml Soluzione Hanks # 4, 10 ml Soluzione Hanks # 5, 0,35 g di sodio bicarbonato, 300 uL di HCl 2M, penicillina-streptomicina 500.000 U) come nel libro Zebrafish (University of Oregon Press, 2000). </li><li> Filo di tungsteno, oro placcato, diametro 0.1mm, Alfa Aesar </li><li> Capillare, diametro 1,0 millimetri, Mondo Strumenti di precisione </li></ol> Parte 4: Procedura di trapianto <ol><li> In una provetta di plastica, preparare 1,2% agarosio a basso punto di fusione di calcio senza ZFR preriscaldato a 40 ° C in un bagno d&#39;acqua. </li><li> Nella fase desiderato (30 HPF, per esempio), prendere gli embrioni in una pipetta Pasteur, lentamente li espellono in provetta, e lentamente li espellono in provetta da centrifuga e incubare per circa 5 secondi. I donatori e gli host devono essere incubate separatamente. </li><li> Con una pipetta, prendere gli embrioni a 1,2% agarosio dalla provetta e disporli su due file parallele in un piatto da 100 millimetri polistirolo Petri, mantenendo gli host e donatori separati. Pipettando fuori lentamente e con attenzione, la maggior parte degli embrioni si trovano su un fianco in agarosio. Avrete bisogno di riorientare quelli che non si trovano in questa posizione utilizzando l&#39;ago smussato in modo che l&#39;occhio è rivolto verso l&#39;alto prima che l&#39;agarosio si solidifica. </li><li> Attendere il 1,2% di agarosio a solidificare, e poi sovrapporre con lo 0,2% soluzione a basso punto di fusione di agarosio. </li><li> Utilizzando l&#39;ago affilato, rimuovere eventuali agarosio intorno agli occhi, e con molta attenzione tagliare la lente fuori dal host e embrioni donatore con piccoli colpi. Assicurati di tagliare solo abbastanza vicino all&#39;obiettivo per non strapparla, ma anche in modo da non rimuovere il tessuto troppo dal resto dell&#39;occhio ospitante. </li><li> Una volta libero, lenti galleggia nel mezzo. Utilizzando l&#39;ago senza punta, con attenzione spingere il donatorelente a poco sopra la posizione in cui l&#39;obiettivo ospitante sarebbe normalmente, e poi spingere verso il basso in un occhio con l&#39;ago smussato. L&#39;obiettivo ospitante può essere scartata. </li><li> Lascia embrioni donatore e ospite in agarosio 1,2% per 30-60 minuti, poi la liberazione dal agarosio utilizzando l&#39;ago appuntito, e trasferirli in un 24-pozzetti con media embrione. Ogni embrione deve occupare una ben separati. Assicurarsi di etichettare i pozzi correttamente in modo che sia chiaro quale host corrisponde a quale donatore. </li><li> Incubare a 28,5 C. Il donatore-derivati ​​obiettivo può essere distinto dalla lente ospitare sul lato non operato dello stesso animale in base a fluorescenza GFP. Le sezioni possono essere fatte anche per misurare le dimensioni dell&#39;obiettivo e per determinare se si differenzia in modo corretto. </li></ol><img src="/files/ftp_upload/1258/1258fig1.jpg" alt="figura 1" /> Fig. 1 immagine a basso ingrandimento di un ago affilato utilizzato per il trapianto di lente. Una capillare di vetro (B) viene inserito nella pipetta Pasteur (A), e un sottile filo di tungsteno (C) viene inserito l&#39;estremità aperta del capillare. Il vetro sopra il filo è ammorbidito con un becco Bunsen. Uso di pinze, tenere ferma la fine di vetro con il filo metallico mentre torcendo l&#39;altra estremità della pipetta Pasteur con la mano. Il vetro dovrebbe ammorbidito a spirale attorno al metallo, presa in posizione. <img src="/files/ftp_upload/1258/fig2 1258.png" alt="" width="600" height="204" /> Fig. 2 Le punte dei due tipi di aghi usati per le lenti trapianto. <img src="/files/ftp_upload/1258/1258fig3.jpg" alt="figura 3" /> Fig. 3 embrioni sottoposti a trapianto di lenti a 30hpf. Vengono mostrati un embrione donatore (A, B), un occhio host con obiettivo trapiantato (C, D), e il lato di controllo dell&#39;embrione ospitante (E, F). Ogni occhio è mostrato in entrambe luce trasmessa e illuminazione UV, come indicato. Fotografie sono state scattate a 48 HPF. Il Q01 [9] linea transgenica è stato utilizzato in questo esperimento.

<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>

Diversi passaggi richiede un&#39;attenzione particolare durante trapianti lente. <ol><li> Aghi affilati: E &#39;meglio preparare gli aghi diversi prima di effettuare il trapianto di lenti, e la punta degli aghi deve essere il più sottile possibile. Un ago più spesso si lacerano il tessuto più a causa del suo diametro più ampio, causando un guasto negli occhi per guarire. </li><li> Organizzare gli embrioni: Giusto prima del trapianto è necessario posizionare gli embrioni in modo che siano coricati su un fianco. ...

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

La lente ha un ruolo importante nello sviluppo della coppa ottica [1,2]. Utilizzando il pesce zebra come organismo modello, le domande riguardanti lo sviluppo dell&#39;obiettivo può essere affrontato. Il pesce zebra è utile per studi di genetica a causa di diverse caratteristiche vantaggiose, tra cui piccole dimensioni, ad alta fecondità, del ciclo di vita breve, e la facilità di cura. Sviluppo lente avviene rapidamente in zebrafish. Da 72 HPF, l&#39;obiettivo zebrafish è funzionalmente maturo [3]. Abbondanti risorse genetiche e molecolari sono a disposizione per sostenere la ricerca in zebrafish. Inoltre, la somiglianza degli occhi zebrafish a quelle di altri vertebrati fornisce basi per il suo uso come un modello animale eccellente di difetti umani [4-7]. Zebrafish mutanti diversi mostrano anomalie lente, tra cui alti livelli di morte cellulare, che in alcuni casi conduce ad una completa degenerazione dei tessuti lente [8]. Per determinare se le anomalie lente sono dovuti a cause intrinseche o difettoso interazioni con i tessuti circostanti, il trapianto di un obiettivo mutante in una wild-type occhio viene eseguita. Utilizzando fuoco lucido aghi di metallo, lenti mutante o wild-type sono accuratamente sezionati dall&#39;animale donatore, e trasferiti nell&#39;ospite. Per distinguere i tessuti wild-type e mutanti, una linea transgenica viene utilizzato come donatore. Questa linea esprime legata alla membrana GFP in tutti i tessuti, compresa la lente. Questa tecnica di trapianto è uno strumento essenziale negli studi di mutanti lente zebrafish.

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

Lente sviluppo comporta interazioni con altri tessuti. Diversi mutanti occhio zebrafish sono caratterizzati da una dimensione lente anormalmente piccole. Qui mostriamo un esperimento di trapianto di lente per determinare se questo fenotipo è dovuto a cause intrinseche o le interazioni difettoso con tessuti che circondano l&#39;obiettivo.

Lens trapianto in Zebrafish e la sua applicazione nell&#39;analisi dei Mutanti Eye

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 Views.  The Second Teaching Hospital of Jilin University. Lente sviluppo comporta interazioni con altri tessuti. Diversi mutanti occhio zebrafish sono caratterizzati da una dimensione lente anormalmente piccole. Qui mostriamo un esperimento di trapianto di lente per determinare se questo fenotipo è dovuto a cause intrinseche o le interazioni difettoso con tessuti che circondano l'obiettivo.

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

Transplantation of Whole Kidney Marrow in Adult Zebrafish

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

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

Il trapianto di midollo renale intero in Zebrafish adulti

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

Ricerca

Biologia

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Il ramo Ladder Walking attività: un sistema di punteggio e la sua applicazione pratica.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

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.

La preparazione e la cultura di espianti Lens Rat epiteliali per lo Studio differenziazione terminale

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.

Cultura primaria e elettroporazione plasmidi dell&#39;Organo murini di 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 ...

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 elettroporazione in vivo dei Morpholinos nella retina Zebrafish per adulti

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 Zebrafish adulti

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.

Progettazione e analisi del comportamento delle preferenze di temperatura e del suo ritmo circadiano 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.

Analisi di Nefrone Composizione e funzione del rene Zebrafish adulti

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.

Sequenziale Applicazione di vetrini di vetro per valutare la compressione Rigidità della lente Mouse: Strain e morfometriche analisi

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.

Ad alta risoluzione Time-lapse imaging e analisi automatizzata dei microtubuli Dynamics in Living ombelicale umano cellule endoteliali Vena

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