Lavoro in laboratorio è supportato da NSF (MCB0919933) e NIH (GM088790).

1. Tronco frammento saggio rigenerazione <ol><li> Smettere di alimentare una coorte di vermi per almeno 5 giorni prima del test rigenerativa per assicurare gli animali sono liberi di rifiuti e di cibo ingerito (~ 30 vermi intatti, ogni verme ~ 8-10 mm lungo post-fame). </li><li> Il giorno del test, lavare un pre-congelato piatto ghiaccio livellato con acqua e coprire la superficie piana ghiacciata con pellicola trasparente. Uso di pinze, mettete una carta da filtro sulla pellicola di plastica. </li><li> Bagnare la carta da filtro con qualche goccia di acqua di sorgente. Trasferimento planarie sul filtro (&lt;20 vermi per filtro) con una pipetta di trasferimento. Worm può essere riposizionato sul filtro, se necessario, repipetting con acqua di sorgente più. Rimuovere il liquido in eccesso. </li><li> Usando un bisturi, amputare la testa da un unico taglio fatto circa metà strada tra l&#39;apice anteriore dell&#39;animale e la fine anteriore della faringe (Figura 1A). </li><li> Usando un bisturi, amputare la regione coda da un unico taglio fatto circa metà strada tra l&#39;estremità della coda dell&#39;animale e la fine posteriore della faringe (Figura 1A). Rimuovere il muco in eccesso sul bisturi con un fazzoletto di carta imbevuto di etanolo al 70% dopo il taglio. Pulire l&#39;etanolo in eccesso dal bisturi. </li><li> Trasferimento del filtro tramite pinze in una capsula di Petri (100 x 25mm profondità) contenente acqua di sorgente. Attendere ~ 3 minuti (per la chiusura della ferita, osservabili da un pizzicotti e oscuramento della ferita). </li><li> Sciacquare frammenti di tronco da carta da filtro nella piastra di Petri contenente supporto desiderato per il dosaggio rigenerativa e trasferimento termostatati incubatore (24 ° C). </li><li> Punteggio fenotipo rigenerativa dopo ~ 1 settimana, quando le strutture rigenerati sono identificabili (Figura 1A). </li></ol> 2. Manipolazione farmacologica della rigenerazione: bipolarismo indotto dal praziquantel <ol><li> Preparare praziquantel (PZQ) stock di farmaci solubilizzanti in DMSO (62.4mg PZQ in 1 ml di magazzino 200mm). Usa il giorno stesso. </li><li> Fai 50mL di droga contenenti soluzione (90 mM PZQ) di sali Montjuch tamponata. Vortex per ~ 1 minuto per garantire la dispersione. </li><li> Eseguire frammento saggio rigenerativa tronco come sopra [protocollo # 1], esponendo una coorte di frammenti di tronco di PZQ al passo finale [1.7]. </li><li> Dopo 24-48 ore, cambio PZQ contenenti supporti per acqua di sorgente, il lavaggio vermi diversi (&gt; 3) volte. </li><li> Ritorna worm per incubatrice. Punteggio bipolarità (a due teste, Figura 1B) una settimana dopo il taglio. </li></ol> 3. Manipolazione genetica di rigenerazione: nel protocollo vivo alimentazione RNAi <ol><li> Prima del dosaggio, la preparazione del fegato omogeneizzato di pollo. Eliminare grassi, le sezioni di colore giallo da foodstock, fegato e purea rimanente. Filtro purea attraverso mesh e sospensione aliquota per la conservazione (1,5 ml tubi, -20 ° C). </li><li> Prima del dosaggio, preparare bovina globuli rossi (RBC) da aliquoting fornitura in campioni di 1 ml, conservati a -20 ° C. </li><li> Seleziona worm per RNAi. Tre coorti sono utilizzati: gene di interesse, controllo positivo (gene noto con fenotipo RNAi; Figura 1C, D &amp; E) e di controllo negativo (gene senza fenotipo RNAi, utile anche per la valutazione dei livelli knockdown rispetto alla coorte sperimentale). Per ciascuna coorte, utilizzare ~ 250 vermi (~ 8-10 mm molto tempo dopo muore di fame per almeno 5 giorni). Conservare in acqua di sorgente in vasca di plastica. </li><li> Per ogni stock disgelo coorte RNAi, di E. coli che esprimono dsRNA gene targeting di interesse [8], insieme ad un tubo di omogenato di fegato di pollo [3.1] e un tubo di GR [3.2]. </li><li> Pellet di batteri (13.000 g, 1 minuto). Rimuovere il surnatante e risospendere pellet in 2xYT media (~ 700μl). Recentrifuge, scartare il surnatante, posto a pellet su ghiaccio. </li><li> Creare un grande foro P200 punta della pipetta, tagliando la fine della punta. Accuratamente risospendere il pellet batterico in una miscela di fegato omogeneizzato di pollo (150 mL) e di globuli rossi (50 mL) per creare mix RNAi di alimentazione. Rimuovere le bolle d&#39;aria per centrifugazione. </li><li> Per l&#39;alimentazione, rimuovere la maggior parte delle acque dalla vasca di plastica contenente vermi (lasciare profondità ~ 1 pollice). Vasca Swirl per concentrare i vermi al centro di questo contenitore, e pipetta mix di alimentazione RNAi in un cerchio corralling vermi. Utilizzare una pipetta di trasferimento di convincere attentamente fuggitivi di nuovo sul mix di alimentazione RNAi senza perturbare altri commensali! </li><li> Dopo l&#39;alimentazione (~ 1 ora), identificare planarie che alimentava bene. Questi worm mostrano una colorazione rosso intenso da globuli rossi ingerito. Scartare gli altri. </li><li> Ricollocare accuratamente soluzione di alimentazione con acqua di sorgente fresca, riducendo al minimo disturbo per impedire egestion di cibo. Per fare questo, delicatamente localizzare i vermi su un lato della provetta con una pipetta di trasferimento, versare l&#39;acqua torbida, e sostituire con acqua fresca versate nella parete opposta della vasca. Planarie Resubmerge velocemente come l&#39;esposizione prolungata all&#39;aria risultati anche in egestion cibo. </li><li> Liberamente séal coperchio del contenitore e tornare alla incubatore (24 ° C) </li><li> Ripetere l&#39;alimentazione protocollo RNAi su più cicli (~ 2-3 giorni di distanza), intervallati da cicli di rigenerazione [protocollo # 1] per lo screening della fenotipi RNAi. Un protocollo standard per l&#39;alimentazione e la rigenerazione efficace per molti geni è mostrato in figura 2, che si ~ 1 mese di durata totale. </li></ol> Modificare questo schema per i geni diversi, come il protocollo ottimale dipenderà da fattori quali la stabilità di mRNA, localizzazione dei tessuti, perdurance proteine, o lo sviluppo di un fenotipo che preclude cicli di alimentazione multipla dopo la rigenerazione. Valutare i livelli di knockdown semplicemente screening per penetranza del fenotipo (se apparente), o da approcci qPCR per confrontare i livelli di mRNA bersaglio con coorte di controllo negativo. 4. Rappresentante dei risultati: Il frammento saggio tronco rigenerazione è robusto in modo che tutti i worm dovrebbe rigenerarsi con le normali antero-posteriore (&#39;testa alla coda&#39;) polarità. Questo può essere segnata appena cinque giorni dopo il taglio, facilitato dalla nascita del ocelli anteriore (asterixed, Figura 1A). Tuttavia, completo ripristino morfologico delle strutture perso si verifica dopo una settimana. Manipolazione farmacologica della rigenerazione tronco frammento da PZQ di cedere a due teste vermi (Figura 1B) è robusto (EC 50 = 87 (+ -) frammenti bipolare 11%, 70μM PZQ per 48 ore 20). Bipolarità avviene su un singolo ciclo rigenerativo. Minore efficacia in questi saggi si riferisce probabilmente a problemi con la solubilità della droga e / o stoccaggio o l&#39;utilizzo di una specie di verme piatto diverso dove PZQ è inefficace. Per i farmaci con la più bassa penetranza, un indice anteriorization è spesso usato per punteggio fenotipi intermedi a parte da 22 bipolarità completa. Fenotipi che derivi dalla RNAi di costrutti di controllo positivo sono mostrati in figura 1: RNAi di Dugesia japonica sei-1 (D j-sei-1) si traduce in un fenotipo senza occhi 23 (Figura 1C), RNAi di Dugesia japonica PC2 (Dj-PC2) traduce in una perdita della risposta avversione luce 24 (Figura 1D) e RNAi di Dugesia japonica βcatenin-1 (Dj-βcatenin-1) si traduce in un due teste fenotipo 16-19, 21 da frammenti tronco (figura 1E). Questi fenotipi possono essere raggiunti utilizzando i seguenti protocolli di RNAi semplice: Dj-sei-1 (FFxFx), Dj-PC2 (FFFx), Dj-βcatenin-1 (FFxFx), dove F = ciclo di alimentazione e x = rispettivamente ciclo rigenerativo. <img src="/files/ftp_upload/3058/3058fig1.jpg" alt="Figura 1"/> Figura 1:... Frammento saggio Tronco rigenerazione A sinistra, immagine di planaria (in alto) e schematica (al centro) per mostrare posizione del frammento asportato tronco diritto, timecourse di rigenerazione del tronco frammento che mostra l&#39;aspetto del frammento rigenerante, a volte indicati (giorni) B immagine di due teste planaria prodotti dall&#39;esposizione PZQ. C verme &#39;Eyeless&#39; prodotta da Dj-sei-1 RNAi. D Perdita di risposta avversione luce in immobile Dj-PC2 RNAi vermi (macchiato rosso), rispetto al verme di controllo mobile (macchiata verde). E bipolare planaria prodotto da Dj-βcatenin-1 RNAi. In BE, la fine originale anteriore del worm RNAi è orientato a sinistra. <img src="/files/ftp_upload/3058/3058fig2.jpg" alt="Figura 2"/> Figura 2: Sequenza di alimentazione e cicli di taglio tipico protocollo per RNAi composto da alimentazioni multiple (F), ed i cicli di rigenerazione (x) che è efficace per atterramento di molti geni in D. japonica. Modifica di questo protocollo per singoli geni possono essere necessari per ottenere risultati ottimali, per esempio utilizzando un numero inferiore (tra parentesi) o un numero maggiore di alimentazione e cicli di rigenerazione.

<ol>
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Nessun conflitto di interessi dichiarati.

I protocolli descritti dettagliatamente i test per lo studio e la manipolazione di rigenerazione della planaria Dugesia japonica. Sono semplici e non richiedono attrezzature specializzate tale da poter essere facilmente eseguito in laboratorio o in aula. Test può essere effettuato singolarmente o combinate (per lo screening genetico chimica dell&#39;efficacia dei farmaci in vivo) e può essere eseguita a livello del gene candidato, o sono adattabili a imparziali, screening ad alto 8. Sia per lo studio della biologia intrigante di planarie in sé, o per la valutazione della funzione in vivo di omologhi mammiferi in un modello alternativo per studiare la rigenerazione dei tessuti, questi approcci dovrebbero catalizzare l&#39;interesse di una vasta gamma di ricercatori.

<table border="1"><tbody><tr><th> Reagente </th><th> Venditore </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Sorgente d&#39;acqua </td><td> Kandiyohi. Premio Waters Inc. Minneapolis, MN </td><td> n / a </td><td> Altre forme di acqua di sorgente funzionano bene anche. Prima prova nei test di vitalità. </td></tr><tr><td> 1 x tamponata Montjuïch sali: NaCl (1,6 mm), CaCl2 (1 mm), MgSO4 (1 mm), MgCl2 (0,1 mm), KCl (0,1 mm), NaHCO3 (1,2 mm), HEPES (1,5 mm). pH 7,4 a 24 ° C. </td><td> Diversi fornitori </td><td> n / a </td><td> Acqua artificiale per trattamenti farmacologici durante i test di rigenerazione per garantire buffer pH. Soluzione 5 / 8 Holtfreter è un&#39;alternativa. </td></tr><tr><td> 2xYT brodo </td><td> Fisher Scientific </td><td> BP2467-500 </td><td> Media = 31 g / L. Sterilizzati in autoclave. </td></tr><tr><td> Petri Dish (100x25mm) </td><td> Fisher Scientific </td><td> 08-757-11 </td><td> Worm alloggio durante i cicli di rigenerazione </td></tr><tr><td> Piazza Dish (100x100x15mm) </td><td> Fisher Scientific </td><td> 08-757-11A </td><td> Riempire con acqua, congelare per il vassoio del ghiaccio utilizzato come superficie di taglio verme </td></tr><tr><td> Vaschetta di plastica: Ziploc Twist &#39;n Loc (16 once). </td><td> Vari rivenditori </td><td> n / a </td><td> Contenitori per l&#39;acqua comoda stretto per coorti RNAi </td></tr><tr><td> Fegato di pollo </td><td> Commerciale di generi alimentari </td><td> n / a </td><td> Propensione fornisce organico, a causa di evitare gli antibiotici. </td></tr><tr><td> Mano Blender </td><td> Ogni cucina fornitore </td><td> n / a </td><td> Utilizzare per le purea di fegato di pollo </td></tr><tr><td> Filo 1 millimetro mesh </td><td> Ogni cucina fornitore </td><td> n / a </td><td> Utilizzare per filtrare purea di fegato di pollo </td></tr><tr><td> Bovina globuli rossi </td><td> Lampire Biological Laboratories </td><td> 7240807 </td><td> 100% lavato e raccolto sospensione RBC </td></tr><tr><td> Filtri circolari </td><td> Whatman # 3 </td><td> 1003 055 </td><td></td></tr><tr><td> Trasferimento Pipettare </td><td> Fisher Scientific </td><td> 13-711-41 </td><td></td></tr><tr><td> Sterili, lame chirurgiche </td><td> I fornitori multipli </td><td> n / a </td><td></td></tr><tr><td> ± Praziquantel </td><td> Sigma Aldrich </td><td> P4668 </td><td> Aliquote polvere Conservare al buio a 4 ° C. Essiccare. </td></tr><tr><td> Dj-sei-1 </td><td></td><td> GenBank AJ557022.1 </td><td> RNAi controllo positivo per fenotipo &quot;occhio-less&quot; 23 </td></tr><tr><td> Dj-βcatenin-1 </td><td></td><td> GenBank AB181913.1 </td><td> RNAi controllo positivo per due teste fenotipo 17 </td></tr><tr><td> Dj-PC2 </td><td></td><td></td><td> RNAi controllo positivo per la perdita del fenotipo avversione leggeri 24 </td></tr></tbody></table>

pharmacological and functional genetic assays to manipulate regeneration of the planarian dugesia japonica

A vita libera planaria vermi piatti hanno una lunga storia di causa dell&#39;utilizzo sperimentale della loro notevoli capacità rigenerative 1. Piccoli frammenti prelevati da questi animali riformare il piano originale corpo dopo la rigenerazione delle strutture del corpo mancanti. Per esempio se frammento di un &#39;tronco&#39; viene tagliato da un worm intatta, la &#39;testa&#39; di un nuovo rigenererà anteriormente e una &#39;coda&#39; rigenererà posteriormente ripristinare l&#39;originaria polarità &#39;testa-coda&#39; delle strutture del corpo prima amputazione (Figura 1A). La rigenerazione è guidato da cellule staminali planaria, noto come &#39;neoblasts&#39;, che si differenziano in circa 30 diversi tipi di cellule normali del corpo durante l&#39;omeostasi e la rigenerazione dei tessuti applicati. Questo processo rigenerativo è robusto e facile da dimostrare. Grazie alla dedizione di alcuni laboratori d&#39;avanguardia, molti strumenti e metodi di genetica funzionale sono stati ottimizzati per questo sistema modello. Di conseguenza, notevoli recenti progressi sono stati fatti nella comprensione e manipolare gli eventi molecolari alla base planaria plasticità dello sviluppo 2-9. Il sistema modello planaria sarà di interesse per una vasta gamma di scienziati. Per i neuroscienziati, il modello offre l&#39;opportunità di studiare la rigenerazione di un intero sistema nervoso, piuttosto che semplicemente la ricrescita / riparazione del singolo processo delle cellule nervose che normalmente sono oggetto di studio in molti modelli consolidati. Planarie esprimere una pletora di neurotrasmettitori 10, rappresentano un importante sistema per lo studio dell&#39;evoluzione del sistema nervoso centrale 11, 12 e hanno un potenziale di screening comportamentale 13, 14. Risultati rigenerativa sono suscettibili di manipolazione da parte apparoaches farmacologiche e genetiche. Per esempio, i farmaci possono essere sottoposti a screening per gli effetti sulla rigenerazione semplicemente inserendo frammenti di corpo in soluzioni contenenti farmaci in diversi momenti dopo l&#39;amputazione. Il ruolo dei singoli geni possono essere studiati utilizzando metodi knockdown (RNAi in vivo), che può essere realizzato attraverso cicli di microiniezione o alimentando batteri-espresso costruisce dsRNA 8, 9, 15. Entrambi gli approcci possono produrre fenotipi di grande impatto visivo ad alta penetranza, ad esempio, la rigenerazione degli animali bipolare 16-21. Per facilitare l&#39;adozione di questo modello e implementazione di tali metodi, che in vetrina protocolli articolo il video per le analisi farmacologiche e genetiche (RNAi in vivo per l&#39;alimentazione) utilizzando il Dugesia planaria japonica.

Watch this Scientific Journal Video about Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica at JoVE.com

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Un modello interessante per studiare la differenziazione delle cellule staminali in un animale vivo è il verme piatto planaria. La rigenerazione è studiato da amputazione esperimenti semplici che sono facili da eseguire in un laboratorio di base e sono suscettibili di (farmacologica e genetica<em&gt; In vivo</em&gt; RNAi) manipolazione come descritto dai protocolli in questo articolo.

I dosaggi farmacologici genetica e funzionale di manipolare Rigenerazione del Planaria Japonica Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

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Biology

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

36.7K Views.  University of Minnesota Medical School. Un modello interessante per studiare la differenziazione delle cellule staminali in un animale vivo è il verme piatto planaria. La rigenerazione è studiato da amputazione esperimenti semplici che sono facili da eseguire in un laboratorio di base e sono suscettibili di (farmacologica e genetica In vivo RNAi) manipolazione come descritto dai protocolli in questo articolo.

Video: Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Luce / buio test di transizione per i Topi

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

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Biologia

Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies

Neurogenesi corticale: Transizione dai progressi nel laboratorio di terapie basate sulle cellule

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Test di induzione neurale nell&#39;embrione Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Elevata Maze Plus per topi

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

Dissezione di giro dentato ippocampale di topo adulto

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Un approccio inverso di test genetici per ridondanza funzionale durante l&#39;embriogenesi

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

Una allele-specifica espressione genica saggio per testare la base funzionale delle Associazioni di genetica

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Planaria immobilizzazione, irradiazione parziale, e di tessuti

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Mappatura batteriche reti funzionali e sulle vie di<em&gt; Escherichia Coli</em&gt; Utilizzando matrici sintetiche genetiche

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

Estratti nucleari di piccole dimensioni per saggi funzionali di espressione genica Macchinari

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...