Ringraziamo la signora Paula Roy per l&#39;allevamento di pesce, e Steve Ekker per il gentile dono del pDB600 plasmide. Questi studi sono stati finanziati da una sovvenzione di SF da NHGRI.

1. Selezione e clonazione di sequenze conservate per i test <ol><li> Si deve prima identificare i potenziali sequenze regolatrici per i test funzionali. Facciamo affidamento sulla conservazione sequenza evolutiva come criterio per selezionare le sequenze, e più spesso usare il PhastCons algoritmo, che è accessibile come una traccia sul browser UCSC Genome (http://genome.ucsc.edu/cgi-bin/hgGateway). Tuttavia, ci sono molti altri algoritmi disponibili per valutare conservazione sequenza tra le specie. </li><li> Una volta che abbiamo selezionato i nostri sequenze, progettiamo primers per amplificare ogni sequenza di DNA genomico. Primers dovrebbero essere selezionati per amplificare la regione conservata più 30 o più coppie di basi su entrambi i lati. </li><li> Per l&#39;amplificazione delle sequenze selezionate usiamo il Takara LA sistema TaqTM. Altre polimerasi funzionerà, ma è importante utilizzare un mix che include un enzima con capacità di correzione di bozze, per ridurre al minimo le possibilità di errori introdotti durante l&#39;amplificazione. Noi verificare la dimensione amplicone con elettroforesi su gel di agarosio. </li><li> Usiamo il pCR 8/GW/TOPO TA Cloning Kit (Invitrogen) per clonare il prodotto di PCR in un vettore di ingresso contenente siti ricombinazione attL, per generare il clone voce di gateway. La reazione clonazione è più efficiente con fresco prodotto di PCR, per cui si consiglia di eseguire la clonazione in un giorno della PCR. Trasformazione in ceppi DH5α viene eseguita seguita dalla selezione resistenza spectinomicina. </li><li> Verifichiamo il prodotto della clonazione TA digestione dei ~ 500ng di plasmide con EcoRI per liberare l&#39;inserto, e confermare la dimensione dell&#39;inserto tramite elettroforesi su gel di agarosio. Si consiglia di sequenziamento per verificare la sequenza di inserimento e di orientamento; primer utilizzati per l&#39;amplificazione può essere utilizzato anche per il sequenziamento. </li><li> Dal clone di ingresso, la non-codificante sequenza è conservata la spola dalla ricombinazione LR al nostro vettore di destinazione Gateway pronto, pGW_cfosGFP1, utilizzando Gateway LR Clonase II enzima Mix (Invitrogen). Trasformazione in ceppi DH5α viene eseguita seguita dalla selezione resistenza all&#39;ampicillina. </li><li> Verifichiamo il prodotto della ricombinazione LR digestione dei ~ 500ng di plasmide con EcoRV per liberare l&#39;inserto, e confermare la dimensione dell&#39;inserto tramite elettroforesi su gel di agarosio. Una volta che un clone preciso è stato identificato, ci prepariamo DNA plasmidico utilizzando il HiSpeed ​​Qiagen ® plasmidi Kit Midi. Abbiamo ulteriormente purificare il plasmide usando la Purificazione QIAquick PCR Kit, secondo protocolli produttore, ed eluire il DNA con 30 microlitri di acqua RNasi libero. Il plasmide è diluito ad una concentrazione di 125 ng / mL e conservato a 4 ° C prima di iniezioni. </li><li> RNA codifica funzionale Tol2 enzima trasposasi è trascritto in vitro dal pDB600 plasmid2. Noi purificare il plasmide con il Midi-Prep kit Qiagen, poi linearizzare 10-20 mg con XbaI. La sintesi di mRNA viene effettuata seguendo il protocollo mMACHINE mMessage Kit (Ambion). </li><li> Noi purificare e precipitare l&#39;RNA secondo le istruzioni del kit. Noi risospendere l&#39;RNA ad una concentrazione finale di circa 1μg/μl, o 20 l per una singola reazione, in acqua RNasi libero, e quantificare mediante spettrofotometria UV. Abbiamo anche analizzare ~ 1μg di RNA mediante elettroforesi su gel di agarosio per verificare la trascrizione completa lunghezza. Anche se un TAE standard o TBE gel sia sufficiente a tale analisi, il buffer campione denaturazione incluso con il kit di trascrizione devono essere utilizzati secondo le istruzioni del kit. </li><li> Ci prova ogni nuovo lotto di trasposasi RNA per l&#39;efficacia eseguendo iniezioni con un noto plasmide (Figura 1). Dopo la verifica, l&#39;RNA è divisa in piccole aliquote e conservati a -80 ° C, per evitare di scongelare e ricongelare ripetuti aliquote individuali. <img src="/files/ftp_upload/1722/1722fig1.jpg" alt="Figura 1" /> Figura 1. Embrione zebrafish iniettato il topo SOX10 enhancer come controllo. (Top) immagine Brightfield (basso) GFP immagine fluorescenza. Ogni nuovo lotto di trasposasi RNA è testato per l&#39;efficacia. </li></ol> 2. Microiniezione di embrioni di zebrafish per l&#39;analisi del mosaico transgenici <ol><li> Tiriamo gli aghi per microiniezione di vetro da 1,2 mm di diametro filamento capillare su un P-97 Sutter Estrattore Micropipetta, con un programma progettato per produrre una punta forte con un cono abbastanza forte da penetrare chorions intatto. Rompiamo le punte a mano allo stereomicroscopio di un diametro esterno di circa 15 micron, con una lama di rasoio pulito e uno scivolo micrometro. Gli aghi può essere fatta il giorno prima di iniezioni, e conservati in un piatto tenendo coperto da pulire. </li><li> Manteniamo il nostro pesce zebra su un normale ciclo di luce scura, con 14 ore di luce, secondo lo standard conditions3. Il giorno prima microiniezioni spettacolo, abbiamo costituito di pesce per accoppiamenti a tempo in serbatoi di piccolo allevamento, ognuno composto da un serbatoio di base, un inserto scanalato, e un coperchio di plastica. Abbiamo posto tre femmine e due maschi per ogni tank in file parallele. </li><li> La mattina del microiniezioni, poco dopo il ciclo di luce comincia, cominciamo combinando maschi e femmine nella pulizia del sistema-acqua trattata per la produzione di uova. E &#39;importante controllare frequentemente il pesce, e raccogliere le uova in pochi minuti di posa, per ottenere lotti ben sincronizzati. Produzione di uova può essere mantenuta per due o tre ore, continuando a combinare gruppi di pesce fresco per tutta la mattina. </li><li> Con un largo foro, 5 1 / 4 &quot;pipetta Pasteur in vetro dotata di un bulbo in lattice, ordiniamo gli embrioni raccolti in 60 x 15 mm Petri, parzialmente riempito con Embryo Medium2, in gruppi di 50 embrioni, e segnare il tempo di raccolti il coperchio di ogni piatto. </li><li> Una volta che i pesci hanno cominciato posa, ci prepariamo soluzione fresca iniezione mescolando quanto segue in una provetta per microcentrifuga sul ghiaccio: <table border="1"><tbody><tr><td> Componente </td><td></td></tr><tr><td> Trasposone magazzino plasmide (125ng/μl) </td><td> 1ml </td></tr><tr><td> Trasposasi RNA stock (175ng/μl) </td><td> 1ml </td></tr><tr><td> Rosso fenolo magazzino (2% in H 2 O) </td><td> 0.5μl </td></tr><tr><td> RNasi libero dell&#39;acqua </td><td> 2.5μl </td></tr></tbody></table> Gli aghi di iniezione sono previste nel tenere piatti e riempito pipettando nl 500 gocce di soluzione di iniezione sull&#39;estremità gamma di ogni ago. Dopo che il liquido viene aspirato alla punta attraverso l&#39;azione capillare, la soluzione è possibile aggiungere a un totale di 1,5-2 microlitri. Prepariamo almeno due aghi per ogni soluzione iniezione. </li><li> L&#39;ago riempito viene caricato nella mano ago titolare di un pneumatico Pico-pompa o simili pressione iniettore, configurato e collegato ad un serbatoio N2 secondo le istruzioni del produttore. Noi calibrare volumi di iniezione misurando il diametro delle goccioline espulse in olio minerale su un vetrino micrometrico. Tipicamente, un tempo di iniezione di 120 ms con una pressione di 20 psi produrrà una goccia di circa 1 nl, ma lievi variazioni nella ago diametro influenzerà questi parametri e di ricalibrazione può essere richiesto tra aghi. </li><li> Le iniezioni vengono effettuate sotto uno stereomicroscopio a 6-10X di ingrandimento. Noi usiamo una pinza sottile per tenere l&#39;embrione in posizione, inserire l&#39;ago di iniezione con una pressione costante attraverso il corion e nel tuorlo di un embrione alla cellula iniziale di una o due stadi cellulari. Idealmente, si posizione la punta dell&#39;ago nel tuorlo appena sotto i blastomeri. Circa 1 nl di soluzione iniettabile viene espulso e l&#39;ago ritirata. Per ciascun costrutto iniettiamo ≥ 200 embrioni. E &#39;anche molto importante, per ogni frizione degli embrioni raccolti, per salvare un piatto di embrioni controllo uninjected. </li><li> Dopo le iniezioni sono stati completati ci ordinare i embrioni eliminando uova non fecondate, embrioni danneggiati, e le iniezioni fallito (embrioni senza rosso fenolo in blastomeri). Abbiamo poi incubare gli embrioni nel medio embrione a 28,5 ° C fino al momento opportuno per le osservazioni. </li></ol> 3. Analisi dei pattern di espressione mosaico <ol><li> A seguito di un tempo di incubazione del caso, esaminiamo gli embrioni iniettati per fluorescenza. La tempistica dipende dal modello normale espressione del gene endogeno zebrafish, ma siamo soliti guardare ogni giorno attraverso i primi 5 giorni dopo la fecondazione. </li><li> Si esaminano gli embrioni per la fluorescenza su un macroscopio Olympus MVX10 misura per epifluorescenza, ma qualsiasi microscopio capace di simili immagini a bassa potenza funzionerà. Se ci sono un gran numero di embrioni che sono morti o sviluppati in modo anomalo il primo giorno, un&#39;occhiata ai controlli uninjected per quella della frizione per vedere se hanno un aspetto normale. Se i controlli appaiono normali, il problema più comune è la purezza insufficiente del DNA iniettato. Inoltre, gli embrioni nelle prime fasi sono sensibili alla quantità totale di plasmide iniettato, quindi è possibile che la quantificazione era impreciso e troppo DNA è stato iniettato. </li><li> Nell&#39;esaminare gli embrioni, di tenere presente che essi saranno tutti mosaico per il costrutto iniettato. Sarà necessario guardare con attenzione tutti gli embrioni, specialmente se il dominio atteso di espressione è di piccole dimensioni. Ricorda inoltre che l&#39;espressione potrebbe essere dinamico; fluorescenza forte il giorno 1 potrebbe scomparire entro il giorno 2, e gli embrioni negativa per l&#39;espressione i primi 3 giorni potrebbe mostrare qualcosa il giorno 4. </li><li> Abbiamo scelto alcuni embrioni con l&#39;espressione più ampia, e utilizzare questi per documentare il modello di fluorescenza da photomicroscopy. Dopo 5 giorni di osservazioni, gli embrioni devono essere restituiti alla struttura e cresciuto come un magazzino. In alcuni mesi, gli adulti possono essere sottoposti a screening per la trasmissione germinale dei transgeni. </li></ol> 4. Risultati Vediamo un ampio variety di modelli e livelli di espressione, a seconda dell&#39;elemento enhancer oggetto di analisi. Di solito siamo molto interessati ad pattern di espressione dei tessuti specifici, e sono spesso concentrandosi su geni espressi nello scheletro. e sono spesso concentrandosi su geni espressi nello scheletro. La Figura 2 mostra esempi di pattern di espressione del mosaico che abbiamo osservato nei nostri embrioni iniettati, con esaltatori di regolazione dell&#39;espressione di cartilagine e ossa. Infine, una parte notevole di sequenze testate non regolano l&#39;espressione specifica del tessuto. Per queste, che più spesso di vedere fluorescenza molto poco, forse poche cellule sparse per embrione. Meno spesso, potremmo vedere la fluorescenza, ma solo in due luoghi comuni di espressione ectopica, l&#39;epidermide e il muscolo scheletrico (Figura 3). <img src="/files/ftp_upload/1722/1722fig2.jpg" alt="Figura 2" /> Figura 2. Esempio di pattern di espressione della cartilagine e dell&#39;osso osservato in embrioni iniettati. <img src="/files/ftp_upload/1722/1722fig3.jpg" alt="Figura 3" /> Figura 3. Non c&#39;è scarsa correlazione tra la posizione del relativo enhancer alla regolazione dei geni e di espressione. Una parte consistente di sequenze testate non regolare l&#39;espressione specifica del tessuto. Questi hanno spesso fluorescenza molto poco, o possono avere due siti comune per l&#39;espressione ectopica: l&#39;epidermide e il muscolo scheletrico. (Top) Brightfield immagine (in basso) GFP immagine fluorescenza.

<ol>
	<li>Fisher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+biological+relevance+of+putative+enhancers+using+Tol2+transposon-mediated+transgenesis+in+zebrafish.">Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish.</a> Nat Protoc. 1 (3), 1297-12 (2006).</li><li>Balciunas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+a+high+cargo-capacity+transposon+for+genetic+applications+in+vertebrates.">Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.</a> PLoS Genet. 2 (11), e169-169 (2006).</li><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. , (1995).</li></ol>

Abbiamo dimostrato l&#39;approccio utilizzato nel nostro laboratorio per l&#39;analisi efficiente di esaltatori di potenziale utilizzo transgenesi zebrafish. Molto spesso, noi usiamo questo metodo per cercare tessuto-specifici associati con esaltatori di geni umani. Nonostante la mancanza di omologia di sequenza evidente maggior parte del tempo tra uomo e pesce sequenze non codificanti, troviamo che questo approccio è di solito efficace nell&#39;identificare esaltatori per i geni di interesse per noi. I fattori critici...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Takara LA Taq</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>RR02M</td>
<td>&nbsp;</td>
<tr>
<td>pCR8/GW/TOPO TA Cloning Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K2500-20</td>
<td>&nbsp;</td>
<tr>
<td>Gateway LR Clonase II enzyme Mix</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11791-100</td>
<td>&nbsp;</td>
<tr>
<td>Library efficiency competent cells DH5&alpha;</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12535-019</td>
<td>&nbsp;</td>
<tr>
<td>Library efficiency competent cells DB3.1</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11782-018</td>
<td>&nbsp;</td>
<tr>
<td>mMESSAGE mMACHINE Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1340</td>
<td>&nbsp;</td>
<tr>
<td>HiSpeed Plasmid Midi Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12643</td>
<td>&nbsp;</td>
<tr>
<td>QIAquick PCR Purification Kit </td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28104</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Pneumatic PicoPump</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>SYS-PV820</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

mosaic zebrafish transgenesis for evaluating enhancer sequences

Il completamento della sequenza del genoma umano, insieme a quello di molte altre specie, ha evidenziato la sfida di attribuire funzione specifica di non sequenze codificanti. Una funzione importante svolta dalla parte non codificante del genoma è di regolare trascrizione del gene, tuttavia, non esistono metodi efficaci per prevedere in generale cis-normativo elementi di sequenza del DNA primario. Abbiamo sviluppato un protocollo efficiente per la valutazione funzionale potenziale cis-normativo elementi attraverso transgenesi zebrafish. Il nostro approccio offre vantaggi significativi rispetto alle tecniche di coltura cellulare a base di geni evolutivamente importante, poiché fornisce informazioni sulla regolazione genica spaziale e temporale. Al contrario, è più veloce e meno costoso di esperimenti simili in topi transgenici, e noi di routine si applica alle sequenze isolate dal genoma umano. Qui mostriamo il nostro approccio alla selezione di elementi per il test basato sulla conservazione di sequenza e il nostro protocollo per le sequenze di clonazione e microinjecting li in embrioni di zebrafish.

Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences at JoVE.com

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

Dimostriamo il nostro approccio alla ricerca di potenziali elementi enhancer regolati da geni evolutivamente e valutando la loro funzione attraverso transgenesi mosaico zebrafish.

Mosaico Transgenesi Zebrafish per la valutazione sequenze Enhancer

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

mosaic-zebrafish-transgenesis-for-evaluating-enhancer-sequences

Research

JoVE Journal

Biology

13.9K Views.  University of Pennsylvania. Dimostriamo il nostro approccio alla ricerca di potenziali elementi enhancer regolati da geni evolutivamente e valutando la loro funzione attraverso transgenesi mosaico zebrafish.

Video: Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafish Cervello iniezione ventricolo

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Ricerca

Biologia

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAi mediata Knockdown Gene e Transgenesi dalla microiniezione nel nematode Necromenic Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

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 ...

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

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

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

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

In elettroporazione in vivo di Morpholinos nella Adult Regenerating Zebrafish Tail Fin

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

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Laser-lesioni del muscolo scheletrico embrionale Zebrafish

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

Il pomodoro / GFP-FLP / FRT Metodo per Live Imaging del Mosaico adulti<em&gt; Drosophila</em&gt; cellule fotorecettrici

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

Separazione di tipi di cellule della spermatogenesi Usando STA-PUT velocità di sedimentazione

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Analisi automatizzata del Dynamic Ca<sup&gt; 2 +</sup&gt; Segnali in sequenze di immagini

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

Un sistema semplificato per la valutazione delle cellule Mechanosensing e Durotaxis<em&gt; In Vitro</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotossina microiniezione in Zebrafish per creare un modello danno renale acuto

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...