Le travail en laboratoire est soutenu par la NSF (MCB0919933) et le NIH (GM088790).

1. Dosage de la régénération Tronc fragment <ol><li> Arrêtez de nourrir une cohorte de vers au moins 5 jours avant le test de régénération pour assurer les animaux sont exempts de déchets et de la nourriture ingérée (~ 30 vers intacte, chaque ver ~ 8-10 mm de long après-faim). </li><li> Le jour du test, rincer un plat pré-gelé glace nivelé avec de l&#39;eau et couvrir la surface plane glacé avec une pellicule de plastique. En utilisant des pinces, placer un papier filtre sur la pellicule de plastique. </li><li> Humidifier le papier filtre avec quelques gouttes d&#39;eau de source. Transfert planaires sur le filtre (&lt;20 vers par filtre) à l&#39;aide d&#39;une pipette de transfert. Les vers peuvent être repositionnées sur le filtre, si nécessaire, par repipetting avec de l&#39;eau plus de ressort. Retirer l&#39;excès de liquide. </li><li> Avec un scalpel, amputer la tête par une seule coupe faite à mi-chemin entre le sommet antérieur de l&#39;animal et l&#39;extrémité antérieure du pharynx (figure 1A). </li><li> Avec un scalpel, amputer la région de la queue par une coupe simple fait environ mi-chemin entre la queue de l&#39;animal et l&#39;extrémité postérieure du pharynx (figure 1A). Retirer l&#39;excès de mucus sur l&#39;utilisation du scalpel une serviette en papier humidifié avec de l&#39;éthanol à 70% après la coupe. Essuyez l&#39;excès d&#39;éthanol à partir de scalpel. </li><li> Transfert filtre via une pince dans une boîte de Pétri (100 mm x 25 mm de profondeur) contenant de l&#39;eau au printemps. Attendre environ 3 minutes (pour fermeture de la plaie, observables par un pincement et assombrissement de la plaie). </li><li> Rincez les fragments du tronc du papier filtre dans une boîte de Pétri contenant du milieu désiré pour l&#39;essai de régénération et de transfert d&#39;incubateur thermostaté (24 ° C). </li><li> Score phénotype régénératrice après ~ 1 semaine, lorsque les structures sont identifiables régénéré (figure 1A). </li></ol> 2. La manipulation pharmacologique de la régénération: bipolarité induite par le praziquantel <ol><li> Préparer le praziquantel (PZQ) des stocks de médicaments par solubilisation dans le DMSO (62.4mg PZQ dans 1mL pour le stock 200mm). Utilisez le même jour. </li><li> Faire 50mL d&#39;une solution contenant des médicaments (90 PZQ uM) en sels Montjuch tamponné. Vortex pour ~ 1 minute pour assurer une dispersion. </li><li> Effectuer test fragment de tronc de régénération tel que détaillé ci-dessus [le protocole n ° 1], exposant une cohorte de fragments de tronc pour PZQ à l&#39;étape finale [1.7]. </li><li> Après 24-48 heures, l&#39;échange PZQ milieux contenant de l&#39;eau au printemps, laver plusieurs vers (&gt; 3) fois. </li><li> Retour vers l&#39;incubateur. Score bipolarité (deux têtes, figure 1B) une semaine après la coupe. </li></ol> 3. La manipulation génétique de la régénération: dans le protocole in vivo d&#39;alimentation ARNi <ol><li> Avant de dosage, de préparer un homogénat de foie de poulet. Jeter gras, jaune sections colorées à partir Foodstock, et le foie de purée restante. Filtre de purée travers un tamis et une suspension pour le stockage aliquote (1,5 ml tubes, -20 ° C). </li><li> Avant de dosage, de préparer bovins globules rouges (hématies) par aliquotage approvisionnement en échantillons de 1 ml, conservés à -20 ° C. </li><li> Sélectionner des vers pour l&#39;ARNi. Trois cohortes sont utilisés: le gène d&#39;intérêt, le contrôle positif (gène avec l&#39;ARNi phénotype est connu; figure 1C, D &amp; E) de contrôle et de négatif (gène sans phénotype ARNi, également utile pour l&#39;évaluation des niveaux de knockdown par rapport à la cohorte expérimentale). Pour chaque cohorte, l&#39;utilisation des vers ~ 250 (~ 8-10 mm de long après avoir faim pendant au moins 5 jours). Conserver dans l&#39;eau de source dans la baignoire en plastique. </li><li> Pour chaque action de cohorte ARNi dégel, de E. coli exprimant le gène d&#39;intérêt ARNdb ciblant [8], avec un tube de l&#39;homogénat de foie de volaille [3,1] et un tube de globules rouges [3,2]. </li><li> Pellet bactéries (13 000 g, 1 minute). Retirer le surnageant et remettre le culot dans 2xYT médias (~ 700μl). Recentrifuger, éliminer le surnageant place, granulés sur la glace. </li><li> Créer un grand alésage P200 pointe de la pipette en coupant l&#39;extrémité de la pointe. Bien resuspendre le culot bactérien dans un mélange de foie de volaille homogénat (150 pi) et les globules rouges (50 pi) de créer des ARNi mélange d&#39;alimentation. Retirer les bulles d&#39;air par centrifugation. </li><li> Pour l&#39;alimentation, retirer la majorité de l&#39;eau de la baignoire en plastique contenant des vers (laisser la profondeur ~ 1 pouce). Swirl baignoire pour se concentrer vers le centre de ce récipient, mélanger la pipette d&#39;alimentation ARNi dans un cercle parcage vers. Utiliser une pipette de transfert soigneusement coaxial évadés en arrière sur le mélange d&#39;alimentation sans perturber l&#39;ARNi autres convives! </li><li> Après l&#39;alimentation (~ 1 heure), identifier les planaires qui ont nourri bien. Ces vers montrent une coloration rouge profond d&#39;érythrocytes ingérés. Jeter les autres. </li><li> Replacez soigneusement l&#39;alimentation solution avec de l&#39;eau de source fraîche, minimisant les perturbations pour empêcher egestion de nourriture. Pour ce faire, doucement localiser les vers d&#39;un côté du tube à l&#39;aide d&#39;une pipette de transfert, versez l&#39;eau trouble, et remplacer l&#39;eau fraîche tombait le mur opposé de la baignoire. Planaires Resubmerge rapidement que l&#39;exposition prolongée à l&#39;air entraîne également egestion alimentaire. </li><li> Librement soicouvercle du récipient et retourner à al incubateur (24 ° C) </li><li> Répétez protocole d&#39;alimentation ARNi sur plusieurs cycles (~ 2-3 jours d&#39;intervalle), entrecoupées de cycles de régénération [protocole n ° 1] à l&#39;écran des phénotypes ARNi. Un protocole standard pour l&#39;alimentation et la régénération efficace pour de nombreux gènes est montré dans la figure 2, ce qui prend environ 1 mois dans la durée totale. </li></ol> Modifier ce régime pour des gènes différents, comme le protocole optimal dépendra de facteurs tels que la stabilité des ARNm, localisation tissulaire, la perdurance de protéines, ou le développement d&#39;un phénotype qui empêche les cycles d&#39;alimentation multiple après régénération. Évaluer les niveaux knockdown simplement le dépistage de la pénétrance du phénotype (si apparente), ou par des approches qPCR pour comparer les niveaux d&#39;ARNm ciblé avec la cohorte de contrôle négatif. 4. Les résultats représentatifs: Le dosage de la régénération du tronc fragment est robuste tel que tous les vers doivent régénérer avec la normale antéro-postérieure («tête à la queue») de polarité. Ceci peut être obtenu dès que cinq jours après la coupe, facilitée par l&#39;émergence de la ocelles antérieure (asterixed, figure 1A). Toutefois, la restauration complète des structures morphologiques perdus survient après une semaine. La manipulation pharmacologique de la régénération fragment de tronc par PZQ pour donner deux vers à tête (figure 1B) est aussi robuste (CE 50 = 87 (+ -) fragments bipolaires 11%, 70μM PZQ pour 48 heures 20). La bipolarité se produit sur un seul cycle de régénération. Moindre efficacité dans ces essais concerne vraisemblablement des problèmes avec la solubilité des médicaments et / ou le stockage ou l&#39;utilisation d&#39;une espèce différente, où plathelminthes PZQ est inefficace. Pour les médicaments à faible pénétrance, un indice anteriorization est souvent utilisé pour marquer phénotypes intermédiaires côté de 22 bipolarité complète. Phénotypes résultant de l&#39;ARNi des constructions de contrôle positifs sont présentés dans la figure 1: l&#39;ARNi des Dugesia japonica six 1 (D j-six-1) se traduit par un phénotype eyeless 23 (figure 1C), d&#39;ARNi Dugesia japonica PC2 (Dj-PC2) en résulte une perte de la réponse aversion pour la lumière 24 (figure 1D) et des ARNi Dugesia japonica βcatenin-1 (Dj-βcatenin-1) se traduit par un phénotype bicéphale 16-19, 21 à partir de fragments du tronc (figure 1E). Ces phénotypes peuvent être obtenus en utilisant les protocoles simples suivantes ARNi: Dj-six-1 (FFxFx), Dj-PC2 (FFFx), Dj-βcatenin-1 (FFxFx), où F = cycle d&#39;alimentation et x = cycle de régénération, respectivement. <img src="/files/ftp_upload/3058/3058fig1.jpg" alt="Figure 1"/> Figure 1:... Test de régénération Tronc fragment A gauche, image de la planaire (en haut) et le schéma (au milieu) pour indiquer l&#39;emplacement du fragment du tronc excisée Droite, timecourse de régénération fragment de tronc montrant l&#39;aspect du fragment régénérant aux temps indiqués (jours) B Image de deux têtes planaire produite par l&#39;exposition PZQ. C &#39;Eyeless «ver produite par Dj-six-1 ARNi. Perte de la réponse de l&#39;aversion D de lumière dans les vers immobiles Dj-PC2 ARNi (coloré en rouge), comparé à un ver de contrôle mobile (tachés vert). E bipolaire planaire produit par Dj-βcatenin-1 ARNi. En BE, la fin originale antérieure du ver ARNi est orientée vers la gauche. <img src="/files/ftp_upload/3058/3058fig2.jpg" alt="Figure 2"/> Figure 2: Séquence d&#39;alimentation et de coupe pour les cycles de RNAi protocole typique comprenant plusieurs tétées (F), et les cycles de régénération (x) qui est efficace pour abattre de nombreux gènes dans D. japonica. La modification de ce protocole pour les gènes individuels peuvent être nécessaires pour obtenir des résultats optimaux, par exemple en utilisant un. Moindre (entre parenthèses) ou un plus grand nombre de cycles d&#39;alimentation et de régénération

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Aucun conflit d&#39;intérêt déclaré.

Les protocoles décrits ici en détail des essais pour étudier et manipuler la régénération de la planaire Dugesia japonica. Elles sont simples et ne nécessitent pas de matériel spécialisé tel qu&#39;ils peuvent être facilement réalisées en laboratoire ou en classe. Les dosages peuvent être effectués individuellement ou combinés (pour le dépistage génétique des produits chimiques des efficacités médicament in vivo) et peut être effectuée au niveau du gène candidat, ou sont adaptables à impartiales, dépistage du débit plus élevé 8. Que ce soit pour étudier la biologie fascinante des Planaires dans leur propre droit, ou pour évaluer la fonction in vivo des homologues de mammifères dans un modèle alternatif pour l&#39;étude de la régénération des tissus, ces approches devraient catalyser l&#39;intérêt d&#39;un large éventail de chercheurs.

<table border="1"><tbody><tr><th> Réactifs </th><th> Vendeur </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> L&#39;eau de source </td><td> Kandiyohi. Prime Waters Inc Minneapolis, MN </td><td> n / a </td><td> D&#39;autres formes d&#39;eau de source bien travailler aussi. Premier essai dans des essais de viabilité. </td></tr><tr><td> 1 x tampon sels Montjuïch: NaCl (1.6mm), CaCl2 (1mm), MgSO4 (1mm), MgCl2 (0,1 mm), KCl (0,1 mm), NaHCO3 (1.2mm), HEPES (1,5 mm). pH 7,4 à 24 ° C. </td><td> Plusieurs fournisseurs </td><td> n / a </td><td> D&#39;eau artificielle pour les traitements médicamenteux au cours des essais de régénération pour assurer tamponnage du pH. Solution de 5 / 8 Holtfreter est une alternative. </td></tr><tr><td> 2xYT Bouillon </td><td> Fisher Scientific </td><td> BP2467-500 </td><td> Médias = 31 g / L. Autoclavés. </td></tr><tr><td> Boîte de Pétri (100x25mm) </td><td> Fisher Scientific </td><td> 08-757-11 </td><td> Vers le logement pendant les cycles de régénération </td></tr><tr><td> Plat carré (100x100x15mm) </td><td> Fisher Scientific </td><td> 08 à 757-11A </td><td> Remplissez avec de l&#39;eau, du gel pour le bac de glace utilisée comme surface de coupe ver </td></tr><tr><td> Baignoire en plastique: n Twist Ziploc »Loc (16 oz). </td><td> Divers détaillants </td><td> n / a </td><td> Récipients d&#39;eau pour les cohortes serrées Pratique ARNi </td></tr><tr><td> Foie de poulet </td><td> Épiceries commerciaux </td><td> n / a </td><td> Biais vers des approvisionnements organiques, en raison de l&#39;évitement d&#39;antibiotiques. </td></tr><tr><td> Mélangeur à main </td><td> Tout fournisseur de cuisine </td><td> n / a </td><td> Utilisez pour faire la purée de foie de volaille </td></tr><tr><td> Fil 1mm mailles </td><td> Tout fournisseur de cuisine </td><td> n / a </td><td> Utilisez pour tamiser la purée de foie de volaille </td></tr><tr><td> Bovine globules rouges </td><td> Lampire laboratoires biologiques </td><td> 7240807 </td><td> 100% lavé et mis en commun la suspension de RBC </td></tr><tr><td> Circulaire papiers filtres </td><td> Whatman n ° 3 </td><td> 1003 055 </td><td></td></tr><tr><td> Pipette de transfert </td><td> Fisher Scientific </td><td> 13-711-41 </td><td></td></tr><tr><td> Stériles, les lames chirurgicales </td><td> Plusieurs fournisseurs </td><td> n / a </td><td></td></tr><tr><td> ± Praziquantel </td><td> Sigma-Aldrich </td><td> P4668 </td><td> Aliquotes Conserver la poudre dans l&#39;obscurité à 4 ° C. Dessécher. </td></tr><tr><td> Dj-six-1 </td><td></td><td> GenBank AJ557022.1 </td><td> ARNi contrôle positif de &quot;eye-moins&quot; 23 phénotype </td></tr><tr><td> Dj-βcatenin-1 </td><td></td><td> GenBank AB181913.1 </td><td> ARNi contrôle positif de deux têtes phénotype 17 </td></tr><tr><td> Dj-PC2 </td><td></td><td></td><td> ARNi contrôle positif pour la perte de l&#39;aversion au phénotype lumière 24 </td></tr></tbody></table>

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

Plathelminthes planaire libre-vivants ont une longue histoire d&#39;utilisation en raison de leurs remarquables expérimentale capacités régénératrices 1. De petits fragments excisés de ces animaux de réforme au plan du corps d&#39;origine après la régénération des structures du corps manquant. Par exemple, si un fragment «tronc» est coupé à partir d&#39;un ver intacts, «tête» d&#39;une nouvelle va régénérer en avant et une «queue» se régénère arrière restaurer l&#39;original de polarité «tête-à-queue» des structures du corps avant l&#39;amputation (figure 1A). La régénération est tirée par les cellules souches planaire, connu comme &#39;néoblastes »qui se différencient en ~ 30 différents types de cellules lors de l&#39;homéostasie normale du corps et la régénération tissulaire appliquée. Ce processus de régénération est robuste et facile à démontrer. Grâce au dévouement de plusieurs laboratoires de pionnier, de nombreux outils et méthodes de génétique fonctionnelle ont été optimisés pour ce système modèle. Par conséquent, des progrès considérables ces dernières ont été réalisés dans la compréhension et la manipulation des événements moléculaires qui sous-tend planaire plasticité développementale 2-9. Le système modèle planaire sera d&#39;intérêt pour un large éventail de scientifiques. Pour les neuroscientifiques, le modèle offre la possibilité d&#39;étudier la régénération d&#39;un système nerveux entier, plutôt que de simplement la repousse / réparation des cellules nerveuses du processus unique qui sont généralement l&#39;objet d&#39;études dans de nombreux modèles établis. Planaires exprimer une multitude de neurotransmetteurs 10, représentent un système important pour étudier l&#39;évolution du système nerveux central 11, 12 et ont un potentiel de dépistage comportementale 13, 14. Régénérative résultats se prêtent à la manipulation par apparoaches pharmacologiques et génétiques. Par exemple, les médicaments peuvent être criblés pour les effets sur la régénération, simplement en plaçant des fragments de corps à la drogue contenant des solutions à différents moments après l&#39;amputation. Le rôle des gènes individuels peuvent être étudiés en utilisant des méthodes knockdown (ARNi in vivo), qui peut être atteint soit par des cycles de micro-injection ou par l&#39;alimentation des bactéries-exprimé construit ARNdb 8, 9, 15. Les deux approches peuvent produire des phénotypes visuellement frappant au-pénétrance de haute, par exemple, la régénération des animaux bipolaires 16-21. Afin de faciliter l&#39;adoption de ce modèle et l&#39;application de telles méthodes, nous présentons dans cet article la vidéo protocoles pour les essais pharmacologiques et génétiques (ARNi in vivo par l&#39;alimentation) en utilisant le Dugesia planaire 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 modèle intéressant pour étudier différenciation des cellules souches au sein d&#39;un animal vivant est le ver plat planaire. La régénération est étudiée par des expériences simples amputations qui sont facilement réalisées dans un laboratoire de base et sont susceptibles d&#39;être (pharmacologiques et génétiques<em&gt; In vivo</em&gt; ARNi) de manipulation tel que détaillé par des protocoles dans cet article.

Pharmacologiques et des essais de génétique pour manipuler la régénération de l&#39;Planaires 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 vues.  University of Minnesota Medical School. Un modèle intéressant pour étudier différenciation des cellules souches au sein d'un animal vivant est le ver plat planaire. La régénération est étudiée par des expériences simples amputations qui sont facilement réalisées dans un laboratoire de base et sont susceptibles d'être (pharmacologiques et génétiques In vivo ARNi) de manipulation tel que détaillé par des protocoles dans cet article.

Vidéo: 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.

Test de transition clair / foncé pour souris

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, ...

Recherche

Biologie

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

Neurogenèse corticale: Passage de progrès dans le laboratoire pour des thérapies cellulaires

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.

Dosage de l&#39;induction neurale dans l&#39;embryon de poulet

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.

Elevated Maze Plus pour Souris

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.

Dissection de l&#39;hippocampe gyrus denté de souris adulte

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.

Une approche de génétique inverse pour tester redondance fonctionnelle pendant l&#39;embryogenèse

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.

Un allèle spécifique Assay expression du gène pour l&#39;épreuve la base fonctionnelle des associations génétiques

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.

Immobilisation planaire, l&#39;irradiation partielle, et la transplantation de tissus

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.

La cartographie des réseaux fonctionnels bactériens et voies de<em&gt; Escherichia Coli</em&gt; En utilisant des tableaux synthétiques génétiques

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.

Les petits extraits nucléaires pour les dosages fonctionnels de l&#39;expression génétique 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 ...