Die Arbeit im Labor ist von der NSF (MCB0919933) und NIH (GM088790) unterstützt.

1. Trunk-Fragment Regeneration Test <ol><li> Stop-Fütterung einer Kohorte von Würmern für mindestens 5 Tage vor der regenerativen Test, um sicherzustellen, Tiere sind frei von Abfall und aufgenommene Nahrung (~ 30 intakt Würmer, jeder Wurm ~ 8-10 mm langen post-Hunger). </li><li> Am Tag des Tests, spülen Sie eine pre-frozen eingeebnet Eis Schüssel mit Wasser und deckt den flachen vereisten Oberfläche mit Klarsichtfolie auslegen. Mit einer Pinzette, stellen Sie einen Filterpapier auf der Plastikfolie. </li><li> Feuchten Sie das Filterpapier mit ein paar Tropfen Quellwasser. Transfer Planarien auf Filterpapier (&lt;20 Würmern pro Filter) mit einem Transferpipette. Worms finden Sie auf Filter neu positioniert werden, falls notwendig, durch repipetting mit mehr Quellwasser. Entfernen Sie überschüssige Flüssigkeit. </li><li> Mit einem Skalpell amputieren den Kopf durch einen einzigen Schnitt etwa auf halbem Weg zwischen der vorderen Spitze des Tieres und dem vorderen Ende des Pharynx (Abbildung 1A) hergestellt. </li><li> Mit einem Skalpell amputieren Heckbereich durch einen einzigen Schnitt gemacht etwa auf halbem Weg zwischen dem hinteren Ende des Tier-und hinteren Ende des Pharynx (Abb. 1A). Entfernen Sie überschüssigen Schleim auf das Skalpell mit einem Papiertuch, befeuchtet mit 70% Ethanol nach dem Schneiden. Überschüssiges Ethanol aus Skalpell. </li><li> Transfer-Filter über Pinzette in eine Petrischale (100 x 25mm Tiefe) mit Quellwasser. Warten Sie ca. 3 Minuten (für den Wundverschluss, beobachtbaren durch einen Quetsch-und Verdunkelung der Wunde). </li><li> Spülen Stamm-Fragmente aus Filterpapier in Petrischale mit gewünschte Medium für regenerative Assay und Transfer zum thermostatisierten Inkubator (24 ° C). </li><li> Ergebnis regenerative Phänotyp nach ~ 1 Woche, wenn regeneriert Strukturen erkennbar (Abb. 1A). </li></ol> 2. Pharmakologische Manipulation der Regeneration: Bipolarität von Praziquantel induzierten <ol><li> Bereiten Sie Praziquantel (PZQ) Lager durch Lösen Medikament in DMSO (62.4mg PZQ in 1ml für 200mm Lager). Verwenden Sie am selben Tag. </li><li> Machen Sie 50 mL der Drogen-haltige Lösung (90 uM PZQ) in gepufferter Montjuch Salze. Vortex für ~ 1 Minute, um die Dispersion zu gewährleisten. </li><li> Führen Stamm-Fragment regenerative Assay wie oben beschrieben [Protokoll Nr. 1], Aussetzen einer Kohorte von Stamm-Fragmente PZQ im letzten Schritt [1,7]. </li><li> Nach 24-48 Stunden, Austausch PZQ-haltigen Medien für Quellwasser, Wasch-Würmer mehrere (&gt; 3) mal. </li><li> Zurück Würmer Inkubator. Ergebnis Bipolarität (zwei Köpfe, Abbildung 1B) 1 Woche nach dem Schneiden. </li></ol> 3. Genetische Manipulation der Regeneration: in vivo RNAi Fütterung Protokoll <ol><li> Vor dem Test vorbereiten Hühnerleber Homogenat. Discard Fettsäuren, gelb gefärbte Abschnitte aus Lebensmittelvorräten und Püree verbleibenden Leber. Filter Püree durch Mesh-Sieb und aliquoten Aufhängung für die Lagerung (1.5 mL Röhrchen, -20 ° C). </li><li> Vor dem Test vorbereiten Rinder roten Blutkörperchen (Erythrozyten) durch Aliquotierung Versorgung in 1ml Proben bei -20 ° C. </li><li> Wählen Würmer für RNAi. Drei Kohorten werden: Gen von Interesse, positive Kontrolle (Gen mit bekannten RNAi-Phänotyp; Abbildung 1C, D &amp; E) und eine negative Kontrolle (Gen ohne RNAi-Phänotyp, auch nützlich für die Beurteilung der Knockdown Ebenen im Vergleich zu experimentellen Kohorte). Für jede Kohorte, verwenden Sie ~ 250 Würmer (~ 8-10 mm lange, nachdem hungern für mindestens 5 Tage). Shop in Quellwasser in Plastikwanne. </li><li> Für jede RNAi Kohorte Tauwetter Lager von E. coli exprimieren dsRNA Targeting Gen von Interesse [8], zusammen mit einem Rohr aus Hühnerleber Homogenat [3.1] und einem Rohr von RBC [3,2]. </li><li> Pellet Bakterien (13.000 g, 1 Minute). Überstand entfernen und Pellet in 2xYT Medien (~ 700μl). Zentrifugieren, Überstand verwerfen, Pellet-Platz auf dem Eis. </li><li> Erstellen Sie eine große Bohrung P200 Pipettenspitze durch das Abschneiden der Ende der Spitze. Gründlich resuspendieren Bakterienpellet in einer Mischung aus Geflügelleber Homogenat (150 mL) und Erythrozyten (50 ul), um RNAi Fütterung Remix erstellt werden. Luftblasen durch Zentrifugation. </li><li> Für die Fütterung, entfernen Sie den größten Teil des Wassers aus der Kunststoffwanne mit Würmern (leave ~ 1 Zoll Tiefe). Swirl Wanne Würmer in der Mitte der Verpackung konzentrieren und Pipette RNAi Fütterung Mischung in einen Kreis einzupferchen Würmer. Verwenden Sie einen Transferpipette sorgfältig Koax-Flüchtlinge zurück auf die RNAi Fütterung Mix ohne störende anderen Gäste! </li><li> Nach der Fütterung (~ 1 Stunde), zu identifizieren Planarien, die gut genährt. Diese Würmer zeigen eine tiefrote Färbung von eingenommenen RBC. Discard andere. </li><li> Vorsichtig wieder Nährlösung mit frischem Quellwasser und minimiert Störungen Egestion von Lebensmitteln zu verhindern. Um dies zu tun, sanft lokalisieren die Würmer auf der einen Seite der Röhre mit einem Transferpipette, gieße trübem Wasser und sorgfältig mit frischem Wasser zu ersetzen ergossen sich die gegenüberliegende Wand der Wanne. Resubmerge Planarien schnell wie bei längerer Exposition an der Luft führt auch in Lebensmitteln Egestion. </li><li> Locker seal Behälterdeckel und Rückkehr zum Inkubator (24 ° C) </li><li> Wiederholen Sie die RNAi Fütterung Protokoll über mehrere Zyklen (~ 2-3 Tage auseinander), mit regenerativen Zyklen [Protokoll Nr. 1], um Bildschirm für RNAi-Phänotypen durchsetzt. Ein Standardprotokoll für die Fütterung und die Regeneration effektiv für viele Gene ist in Abbildung 2, die ca. 1 Monat dauert in Gesamtdauer angezeigt. </li></ol> Ändern Sie diese Regelung für verschiedene Gene, wie eine optimale Protokoll auf Faktoren wie die Stabilität der mRNA-, Gewebe-Lokalisierungs-, Protein-perdurance oder die Entwicklung von einem Phänotyp, der mehrere Werkstücksumspannzyklen schließt nach der Regeneration abhängen wird. Beurteilen knockdown Ebenen durch einfaches Screening nach Penetranz des Phänotyps (wenn erkennbar), oder durch qPCR Ansätze zur gezielten mRNA-Spiegel mit negativen Kontrolle Kohorte zu vergleichen. 4. Repräsentative Ergebnisse: Der Stamm-Fragment Regeneration Test ist robust, so dass alle Würmer sollten mit normaler anterior-posterior (&quot;Kopf-Schwanz&quot;) Polarität zu regenerieren. Dies kann, sobald 5 Tage nach dem Schneiden, durch das Aufkommen des vorderen Augenflecken (asterixed, Abbildung 1A) erleichtert gewertet. Allerdings tritt vollständige morphologische Wiederherstellung der verlorenen Strukturen nach einer Woche. Pharmakologische Manipulation von Stamm-Fragment Regeneration durch PZQ zu zweiköpfigen Würmer Ausbeute (Abbildung 1B) ist auch robust (EC 50 = 87 (+ -) 11% bipolare Fragmente, 70μM PZQ für 48 Stunden 20). Bipolarität erfolgt über eine einzige regenerative Zyklus. Geringere Wirksamkeit bei diesen Tests wahrscheinlich bezieht sich auf Probleme mit Drogen Löslichkeit und / oder Lagerung oder Verwendung eines anderen flatworm Arten, bei denen PZQ ist unwirksam. Bei Medikamenten mit geringer Penetranz, ist eine Vorverlegung Index häufig verwendet, um Zwischen-Phänotypen beiseite Ergebnis aus kompletten Bipolarität 22. Phänotypen, die sich aus RNAi der positiven Kontrolle Konstrukte sind in Abbildung 1 dargestellt: RNAi von Dugesia japonica sechs 1 (D j-six-1) führt zu einer eyeless Phänotyp 23 (1C), RNAi von Dugesia japonica PC2 (Dj-PC2) führt zu einem Verlust des Lichtes Abneigung Antwort 24 (1D) und RNAi von Dugesia japonica βcatenin-1 (Dj-βcatenin-1) führt zu einer zweiköpfigen Phänotyp 16-19, 21 aus Stamm-Fragmente (Abb. 1E). Diese Phänotypen lassen sich mit den folgenden einfachen RNAi Protokolle werden: Dj-six-1 (FFxFx), Dj-PC2 (FFFx), Dj-βcatenin-1 (FFxFx), wo F = Fütterung Zyklus und x = regenerative Zyklus jeweils. <img src="/files/ftp_upload/3058/3058fig1.jpg" alt="Abbildung 1"/> Abbildung 1:... Trunk Fragment Regeneration Assay A Left, Image der Planarie (oben) und schematische (Mitte) auf Position herausgeschnitten Stamm-Fragment zeigen Right, zeigt Zeitverlauf der Stamm-Fragment Regeneration Aussehen zu regenerieren Fragment bei angegebenen Zeiten (Tage) B Image of zweiköpfigen Planarie durch PZQ Belichtung hergestellt. C &#39;Eyeless &quot;Wurm von Dj-six-1 RNAi. D Lichtverlust Abneigung Reaktion in immobile Dj-PC2 RNAi Würmer (rot gefärbt), im Vergleich zu mobilen Kontrolle Wurm (gebeizt hergestellt grün). E Bipolar Planarie von Dj-βcatenin-1 RNAi produziert. In BE, ist die ursprüngliche vordere Ende des RNAi Würmer orientiert sich an der linken Seite. <img src="/files/ftp_upload/3058/3058fig2.jpg" alt="Abbildung 2"/> Abbildung 2: Ablauf der Fütterung und Schneiden Zyklen für typische RNAi-Protokoll aus mehreren Fütterungen (F), und der regenerativen Zyklen (x), die effektiv für Knockdown von vielen Genen in D. ist japonica. Änderung dieses Protokolls für einzelne Gene erforderlich sein, um optimale Ergebnisse zu erzielen, zum Beispiel mit einem weniger (Klammern) oder eine größere Anzahl von Fütterung und regenerative Zyklen.

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Die Protokolle hier beschriebenen Details Assays zur Untersuchung und Manipulation Regeneration der Planarien Dugesia japonica. Sie sind einfach und erfordern keine spezielle Ausrüstung, so dass sie leicht im Labor oder im Klassenzimmer durchgeführt werden. Assays können einzeln ausgeführt werden, oder kombiniert (für chemisch-genetischen Screening von Drogen Wirkungsgraden in vivo) und kann bei der Kandidaten-Gen-Ebene durchgeführt werden, oder sind anpassungsfähig, unvoreingenommene, höheren Durchsatz-Screening 8. Ob für das Studium der faszinierenden Biologie der Planarien in ihrem eigenen Recht oder die Bewertung der in vivo-Funktion von Säuger-Homologen in einem alternativen Modell zum Studium der Regeneration der Gewebe, sollten diese Ansätze Interesse aus einem vielfältigen Angebot von Forschern zu katalysieren.

<table border="1"><tbody><tr><th> Reagens </th><th> Verkäufer </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> Quellwasser </td><td> Kandiyohi. Premium-Waters Inc. Minneapolis, MN </td><td> n / a </td><td> Andere Formen von Quellwasser gut funktionieren auch. Studie erstmals Lebensfähigkeit Assays. </td></tr><tr><td> 1 x gepufferte Montjuïch Salze: NaCl (1,6 mm), CaCl2 (1mm), MgSO4 (1mm), MgCl2 (0,1 mm), KCl (0,1 mm), NaHCO3 (1,2 mm), HEPES (1,5 mm). pH 7,4 bei 24 ° C. </td><td> Mehrere Anbieter </td><td> n / a </td><td> Künstliche Wasser für medikamentöse Behandlungen während regenerative Assays zur pH-Pufferung zu gewährleisten. 5 / 8 Holtfreter-Lösung ist eine Alternative. </td></tr><tr><td> 2xYT Broth </td><td> Fisher Scientific </td><td> BP2467-500 </td><td> Media = 31 g / L. Autoklaviert. </td></tr><tr><td> Petrischale (100x25mm) </td><td> Fisher Scientific </td><td> 08-757-11 </td><td> Housing Würmer während Regenerationszyklen </td></tr><tr><td> Platz Dish (100x100x15mm) </td><td> Fisher Scientific </td><td> 08 bis 757-11A </td><td> Füllen Sie mit Wasser, einfrieren für Eiswürfelschale als Wurm Schnittfläche verwendet </td></tr><tr><td> Kunststoffwanne: Ziploc Twist &#39;n Loc (16oz). </td><td> Verschiedene Einzelhändler </td><td> n / a </td><td> Komfortable wasserdichte Behälter für RNAi Kohorten </td></tr><tr><td> Geflügelleber </td><td> Handelsregister Lebensmittelgeschäfte </td><td> n / a </td><td> Bias in Richtung Bio-Lieferungen wegen Vermeidung von Antibiotika. </td></tr><tr><td> Stabmixer </td><td> Jede Küche Lieferanten </td><td> n / a </td><td> Verwenden Sie für die Herstellung Hühnerleber Püree </td></tr><tr><td> Draht 1mm Mesh-Sieb </td><td> Jede Küche Lieferanten </td><td> n / a </td><td> Verwenden Sie zum Spannen Hühnerleber Püree </td></tr><tr><td> Bovine roten Blutkörperchen </td><td> Lampire Biological Laboratories </td><td> 7240807 </td><td> 100% gewaschene und RBC-Suspension gebündelt </td></tr><tr><td> Circular Filterpapiere </td><td> Whatman Nr. 3 </td><td> 1003 055 </td><td></td></tr><tr><td> Transferpipette </td><td> Fisher Scientific </td><td> 13-711-41 </td><td></td></tr><tr><td> Sterile, chirurgische Messer </td><td> Multiple Vendors </td><td> n / a </td><td></td></tr><tr><td> ± Praziquantel </td><td> Sigma Aldrich </td><td> P4668 </td><td> Shop Pulver Aliquots im Dunkeln bei 4 ° C. Austrocknen. </td></tr><tr><td> Dj-six-1 </td><td></td><td> GenBank AJ557022.1 </td><td> RNAi positive Kontrolle für &quot;eye-less&quot;-Phänotyp 23 </td></tr><tr><td> Dj-βcatenin-1 </td><td></td><td> GenBank AB181913.1 </td><td> RNAi positive Kontrolle für zweiköpfige Phänotyp 17 </td></tr><tr><td> Dj-PC2 </td><td></td><td></td><td> RNAi positive Kontrolle für den Verlust von Licht Abneigung Phänotyp 24 </td></tr></tbody></table>

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

Frei lebende Plattwürmer Planarien haben eine lange Geschichte der experimentellen Nutzung aufgrund ihrer bemerkenswerten Regenerationsfähigkeit 1. Kleine Fragmente von diesen Tieren herausgeschnitten Reform der ursprüngliche Bauplan folgende Regeneration der fehlenden Strukturen des Körpers. Zum Beispiel, wenn ein &quot;Stamm&quot;-Fragment aus einer intakten Wurm geschnitten wird, einen neuen &quot;Kopf&quot; wird nach vorne zu regenerieren und einen &quot;Schwanz&quot; wird hinten regenerieren Wiederherstellung des ursprünglichen &quot;Kopf-an-Schwanz&quot; Polarität von Körperstrukturen vor der Amputation (Abbildung 1A). Die Regeneration wird durch Planarien Stammzellen als &quot;Neoblasten&quot;, die in ~ 30 verschiedene Zelltypen differenzieren während der normalen Homöostase des Körpers und durchgesetzt Geweberegeneration bekannt angetrieben. Diese regenerative Prozess ist robust und leicht zu demonstrieren. Durch das Engagement mehrerer bahnbrechender Labs, viele Werkzeuge und funktionellen genetischen Methoden haben nun für dieses Modell-System optimiert. Folglich hat erhebliche jüngsten Fortschritte im Verständnis und die Manipulation der molekularen Ereignisse zugrunde Planarie Entwicklungsstörungen Plastizität 2-9 worden. Die Planarie Modellsystem von Interesse, eine breite Palette von Wissenschaftlern werden. Für Neurowissenschaftler, bietet das Modell die Möglichkeit, die Regeneration einer ganzen Nervensystems Studie, anstatt einfach nur das Nachwachsen / Reparatur von einzelnen Nervenzelle Prozess, der in der Regel stehen im Mittelpunkt der Studie in vielen etablierten Modellen. Planarien Ausdruck einer Vielzahl von Neurotransmittern 10, sind ein wichtiger für die Erforschung von Evolution des zentralen Nervensystems 11, 12 und Verhaltensstörungen Screening potenzieller 13, 14. Regenerative Ergebnisse sind zugänglich für Manipulation durch pharmakologische und genetische apparoaches. Zum Beispiel können Medikamente auf ihre Wirkung auf die Regeneration einfach, indem man Körper-Fragmenten in Drogen-haltige Lösungen zu verschiedenen Zeitpunkten nach der Amputation zu sehen sein. Die Rolle einzelner Gene kann studierte mit Knockdown-Methoden (in vivo RNAi), die entweder durch Zyklen von Mikroinjektion oder durch Fütterung dsRNA-Konstrukte 8, 9, 15 bakteriell exprimiert erreicht werden kann. Beide Ansätze können optisch ansprechende Phänotypen mit hoher Penetranz, zum Beispiel die Regeneration von bipolaren Tiere 16-21 zu produzieren. Zur Erleichterung der Einführung dieses Modells und Umsetzung solcher Methoden, präsentieren wir in diesem Video Artikel Protokolle für pharmakologische und genetische Tests (in vivo RNAi durch Füttern) mit der Planarie Dugesia 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

Ein attraktives Modell zur Untersuchung Stammzell-Differenzierung innerhalb eines lebenden Tieres ist der Planarie flatworm. Die Regeneration wird durch einfache Amputation Experimente, die leicht in einem der Grundlagenforschung im Labor durchgeführt werden untersucht und sind zugänglich für pharmakologische und genetische (<em&gt; In vivo</em&gt; RNAi) Manipulation durch Protokolle in diesem Artikel beschrieben.

Pharmakologische und Functional genetischen Tests zu manipulieren Regeneration der Planarien Dugesia japonica</em

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

pharmacological-functional-genetic-assays-to-manipulate-regeneration

Research

JoVE Journal

Biology

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

36.7K Aufrufe.  University of Minnesota Medical School. Ein attraktives Modell zur Untersuchung Stammzell-Differenzierung innerhalb eines lebenden Tieres ist der Planarie flatworm. Die Regeneration wird durch einfache Amputation Experimente, die leicht in einem der Grundlagenforschung im Labor durchgeführt werden untersucht und sind zugänglich für pharmakologische und genetische ( In vivo RNAi) Manipulation durch Protokolle in diesem Artikel beschrieben.

Serie: 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.

Hell / Dunkel Transition Test für Mäuse

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, ...

Forschung

Biologie

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

Kortikale Neurogenese: Übergang von Advances in das Labor zu zell-basierte Therapien

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.

Assay für Neural Induction im Hühnerembryo

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 Plus Maze für Mäuse

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 der hippocampalen von erwachsenen Maus

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.

Ein Reverse Genetic Ansatz zur funktionellen Redundanz während der Embryogenese-Test

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.

Ein Allel-spezifische Gene Expression Assay zur Funktionsanalyse Grundlage genetischer Verbände testen

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.

Planarien Immobilisierung, partielle Bestrahlung, und Gewebetransplantation

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.

Mapping Bakterielle Functional Netzwerke und Pathways in<em&gt; Escherichia Coli</em&gt; Verwendung von 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 ...

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.

Small-scale Kernextrakten für funktionelle Assays von Gen-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 ...