Dieses Protokoll wird derzeit in unserem Labor eingesetzt und hat im Laufe der Jahre von vielen Menschen, darunter Dr. David Sharp, Mijung Kwon, Patrizia Sommi, Dhanya Cheerambathur verfeinert. Wir danken Dr. Bill Sullivan (UCSC), die uns mit ausgezeichneten Beratung auf die Manipulation und Mikroinjektion der frühen Drosophila-Embryonen, wenn unsere Arbeit in diesem System, das initiiert wurde (Code 13). Wir danken allen Mitgliedern des Scholey Labor. Unsere Arbeit auf die Mitose in Drosophila wird durch NIH GM55507 unterstützt.

Rezepte: Grape Juice Platten: <ul><li> 5.5g Bacto-Agar </li><li> 14,5 g Dextrose oder Glucose </li><li> 7,15 g Saccharose </li><li> 45 ml Traubensaft-Konzentrat (100% Saft). </li><li> 204,5 ml H 2 0 </li><li> 625 ul 10N NaOH </li></ul> Alle Zutaten mischen und eine Mikrowelle zum Kochen bringen. Fügen Sie 2,8 ml saure Mischung (Säure-Mix: 20,9 ml Propionsäure, 2,1 ml Phosphorsäure, 27 ml H 2 0) Mix und gießen auf 35 mm Petrischalen. Lassen Sie erstarren bei Raumtemperatur für ein oder zwei Tage. Wenn die Platten sind nicht zu früh eingesetzt werden, mit Parafilm versiegeln und halten bei 4 ° C (allow auf RT, bevor Sie ins Gleichgewicht). Hefepaste: Lösen Sie etwa einen Teelöffel Hefe (Sigma YSC2, Hefe aus Saccharomyces cerevisiae Typ II) in Wasser zu einer dicken Paste. Legen Sie eine kleine Menge davon auf jeden Traubensaft Platte kurz vor dem Gebrauch. G-PEM-Puffer: <ul><li> 80 mM Na-Pipes, pH 6,9 </li><li> 1 mM MgCl 2 </li><li> 1 mM EGTA </li><li> 1 mM GTP </li></ul> Injektionspuffer: <ul><li> 150 mM K-Aspartat </li><li> 10 mM K-Phosphat- </li><li> 20 mM Imidazol, pH 7,2 </li></ul> Heptan Kleber: Rollen Sie doppelte Klebeband und in eine 100ml Flasche, ca. 50ml Heptan hinzu, verschließen Sie die Flasche, und Rock für mehrere Tage. Bei der Verwendung von, fügen Heptan, wenn der Kleber ist zu dick. Dehydration Kammer: Werfen Sie einen 100 mm Petrischale, legte ein Teil einer 35-mm-Schale im Inneren zu einem &quot;table&quot; zu machen und stell Drierite (wasserfreies Calciumsulfat) um ihn herum, so dass die Höhe der Drierite nicht höher ist als der &quot;Tisch&quot; und Deckel. Das Deckglas mit Embryonen werden auf dieser &quot;Tabelle&quot; zur Entwässerung werden vor der Injektion gelegt. Es ist eine gute Idee, zumindest einige der angibt, Drierite verwenden und ändern Sie es, wenn es Farbe hat sich geändert. Protokoll: Dieses Protokoll kann die Injektion von praktisch jeder lösliche Reagenz in die Drosophila-Syncytial-Embryo eingesetzt werden: Zum Beispiel, entweder ein fluoreszierendes Protein zur Beobachtung oder ein Zielprotein Hemmer oder beides. Getting alles bereit: 1 .- 2 bis 3 Tage vor Experiment machen Traubensaft Platten und richten Sie legen Käfig: Nimm eine Plastikflasche (6 oz, runder Boden von Applied Scientific Drosophila), schneiden Sie zwei kleine Löcher (ca. 1cm 2) auf gegenüberliegenden Seiten des Flasche füllen und mit einem Baumwolltuch Stück (dies wird damit die Luft flow), tippen Sie auf die Fliegen in die Flasche geben und mit einem Traubensaft Platte. Halten bei 25 ° C (oder Raumtemperatur), sollten fliegt Embryonen für etwa 10 Tage liegen. 2 .- Mindestens ein Tag vor dem Experiment von Nadeln zur Injektion ziehen, verwenden wir einen Kopf Nadel / Pipette Puller Modell 720 und dünnwandigen Glasfasern (World Precision Instruments tw100f). Für die Injektion von fluoreszierenden Tubulin, halten Nadeln bei 4 ° C auf Temperatur-induzierte Polymerisation in der Nadel zu verhindern. Tubulin Vorbereitung: Hinweis: Alle arbeiten mit Tubulin sollte bei 4 ° C, um die Polymerisation zu verhindern. <ol><li> Nehmen gekennzeichnet Tubulin aus der Tiefkühltruhe und legte es auf Eis für 5 bis 15 Minuten. </li><li> Verdünnen mit G-PEM-Puffer (4 bis 10 fach je nach Intensität erforderlich). </li><li> Zentrifuge bei 13k rpm bei 4 ° C für 10-15 Minuten. </li><li> Legen Sie 1 bis 2 ul in Nadel. </li></ol> Antikörper oder chemische Inhibitor: Bereiten Nadeln für Tubulin, mit einer geeigneten Konzentration (dies kann geprüft werden müssen). Der Antikörper kann in Injektionspuffer werden, G-PEM-Puffer oder PBS. Wenn ein anderer Puffer erforderlich ist, wird dieser Puffer müssen als Kontrolle injiziert werden, um sicherzustellen, dass dieser Puffer verursacht keine Schäden am eigenen. Hinweis: Inhibitor und fluoreszierende Tubulin können gemischt und gemeinsam, wenn die Konzentrationen es ermöglichen injiziert. Ansonsten injizieren das fluoreszierende Tubulin und warten 5 bis 10 Minuten vor Injektion des Inhibitors. Bei der Durchführung einer doppelten Einspritzung, mit mehr Embryonen zu starten, da weniger Embryonen in der Regel erholen wird. Embryo-Entnahme: <ol><li> Legen Sie eine neue Traubensaft Platte auf die Gestaltung Käfig. </li><li> Entfernen Sie diese Platte nach einer Stunde, das ist die erste Kollektion des Tages und oft nicht sehr gut. Es kann verworfen werden. </li><li> Ändern Sie den Teller pro Stunde zu sammeln Embryonen für jeweils eine Stunde zu halten. </li></ol> Deckglas Zubereitung: <ol><li> Setzen Sie ein 50 x 22mm Deckglas auf der einen Seite einen Objektträger. Kleben Sie die vier Ecken, um die Folie, damit es nicht zu bewegen. </li><li> Mit einem Wattestäbchen, legte eine Schicht aus Heptan Leim in einer Zeile auf dem Deckglas (der Kleber sollte nicht viskos und sollte in ein paar Sekunden trocknen, sonst fügen Sie mehr Heptan). </li><li> Legen Sie ein StückDoppel-Klebeband auf der Folie auf der Seite des Deckglases. </li></ol> Embryo Zubereitung: Hinweis: Die Embryonen etwa 2 Stunden nach Beginn der Sammlung abgebildet werden sollen, so beginnen die folgenden Schritte ausreichend Zeit, um sie innerhalb dieses Zeitrahmens zu beenden. <ol><li> Mit einem feuchten Pinsel vorsichtig abholen Embryonen aus dem Traubensaft Platte und am Doppel-Klebeband auf dem Objektträger. </li><li> Mit den äußeren Teil der Pinzette, rollen die Embryonen in den Doppel-Klebeband bis zum Chorion (die äußere Membran) bricht. </li><li> Pick-up der Embryo durch ihn sanft über das Chorion dass es auf die Pinzette Stöcken und legen Sie es auf dem Heptan Kleber auf das Deckglas mit der langen Seite des Embryos parallel zur langen Seite des Deckglases. Set up von 10 bis 20 Embryonen in einer Zeile. </li><li> Entfernen Sie das Deckglas und legen Sie sie in die Dehydratisierung Kammer für 3 bis 8 Minuten (dies hängt von der Luftfeuchtigkeit des Raumes und der Menge, die injiziert werden). </li><li> Auf ein Metall-Kammer mit Vakuum-Fett. </li><li> Decken Sie die Embryonen mit Halocarbonöl 700 bis Vermeidung weiterer Austrocknung. Embryonen sind nun bereit für die Injektion. </li></ol> Embryo-Injektion: <ol><li> Finden Sie die Embryonen unter einem 16x Objektiv. </li><li> Bewegen Sie den Embryonen entfernt, und finden Sie die Nadel ohne Verschieben der Fokusebene. </li><li> Zentrieren Sie die Nadel aus und verschieben ihn, ohne es in x oder y-Richtung. </li><li> Wenn die Nadel nicht geöffnet ist, legen Sie die Kante des Deckglases in das Blickfeld, aber nicht dort, wo die Nadel sein wird, und senken Sie die Nadel auf die gleiche Bildebene. Sehr vorsichtig bewegen das Deckglas, bis sie die Nadel Hits und sanft bricht sie zu öffnen. Wenn die Nadel zu öffnen, gehen Sie zu Schritt 5 fort. (Nadeln können mit Flusssäure vor der Befüllung geöffnet werden). </li><li> Legen Sie die Embryonen in Aussicht (aber nicht, wo die Nadel wird), senken Sie die Nadel in die Öl-und vergewissern Sie erhalten schöne Flüssigkeitstropfen von der Nadel. </li><li> Vorsichtig, aber stetig bewegen den Embryo in die Nadel und Injektion ein Tropfen in den Embryo und bewegen Sie den Embryo entfernt. (Oder folgen Sie den Anweisungen Ihres Injektionsgeräts). </li><li> Schließlich wurden die Embryonen injiziert worden, sind sie bereit für die Beobachtung an einem konfokalen Mikroskop. </li></ol> Imaging: Bild der Embryonen auf einem konfokalen Mikroskop mit einer 60 oder 100X Objektiv: Notes: <ol><li> Der zeitliche Abstand auf einer Zeitserie sollte höchstens ein paar Sekunden, da die Mitose in der Embryo ist sehr schnell, je nach den genauen Ablauf zu untersuchen. </li><li> Eine 3D-Serie oder eine einzelne Ebene kann je nach Verfahren von Interesse erworben werden. </li><li> Für schnell wirkende Inhibitoren, hat die Übertragung von Injektion Mikroskop Imaging-Mikroskop, schnell zu sein. </li></ol><img src="/files/ftp_upload/1382/1382fig1.jpg" alt="Abbildung 1" /> Abbildung 1: Studium der Mitose in der Drosophila-Syncytial-Embryo. Kernen im frühen Embryo zu unterziehen schnellen Divisionen ohne Zytokinese, während der Zyklen 10 bis 13 Kerne bilden eine Monoschicht an der Hirnrinde. A. Schematische Darstellung des frühen Embryos mit Kernen bei Cortex. Embryo ausdrücken können GFP und / oder RFP markierten Proteine ​​und kann mit anderen markierten Proteine ​​und / oder Inhibitoren mikroinjiziert werden. B. Zeitraffer-Aufnahme von Embryos, die GFP-Tubulin-und RFP-Histon. C. Plot des Pol-Pol-Trennung für Fahrräder 11 , 12 und 13 in einem Wildtyp-Embryo. Spindellänge Exponate Perioden der isometrischen Länge und Perioden der Dehnung, die sehr konstant und reproduzierbar sind. D. Dynamik der Spindel Mikrotubuli. Die Injektion von geringen Konzentration von Rhodamin Tubulin führt zu Flecken von wenigen Tubulin-Untereinheiten, die verwendet werden, um die Dynamik der Mikrotubuli-Studie kann gebildet.

<ol>
	<li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+dynamics+in+Drosophila.">Mitotic spindle dynamics in Drosophila.</a> Int Rev Cytol. 259, 139-172 (2007).</li><li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4+system+in+Drosophila:+a+fly+geneticist's+Swiss+army+knife.">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. 34, 1-15 (2002).</li><li>Morris, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+methods+for+analyzing+the+functions+of+kinesins+in+early+embryos.">Microinjection methods for analyzing the functions of kinesins in early embryos.</a> Methods Mol Biol. 164, 163-172 (2001).</li><li>Brust-Mascher, I., Sommi, P., Cheerambathur, D. K., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin-5-dependent+poleward+flux+and+spindle+length+control+in+Drosophila+embryo+mitosis.">Kinesin-5-dependent poleward flux and spindle length control in Drosophila embryo mitosis.</a> Mol Biol Cell. 20, 1749-1762 (2009).</li><li>Brust-Mascher, I., Civelekoglu-Scholey, G., Kwon, M., Mogilner, A., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+for+anaphase+B:+role+of+three+mitotic+motors+in+a+switch+from+poleward+flux+to+spindle+elongation.">Model for anaphase B: role of three mitotic motors in a switch from poleward flux to spindle elongation.</a> Proc Natl Acad Sci U S A. 101, 15938-15943 (2004).</li><li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+flux+and+sliding+in+mitotic+spindles+of+Drosophila+embryos.">Microtubule flux and sliding in mitotic spindles of Drosophila embryos.</a> Mol Biol Cell. 13, 3967-3975 (2002).</li><li>Cheerambathur, D. K., Brust-Mascher, I., Civelekoglu-Scholey, G., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+partitioning+of+mitotic+kinesin-5+crosslinkers+between+microtubule-bound+and+freely+diffusing+states.">Dynamic partitioning of mitotic kinesin-5 crosslinkers between microtubule-bound and freely diffusing states.</a> J Cell Biol. 182, 421-428 (2008).</li><li>Cheerambathur, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+an+anaphase+B+switch:+predicted+role+for+a+microtubule+catastrophe+gradient.">Quantitative analysis of an anaphase B switch: predicted role for a microtubule catastrophe gradient.</a> J Cell Biol. 177, 995-1004 (2007).</li><li>Kwon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromokinesin,+KLP3A,+dives+mitotic+spindle+pole+separation+during+prometaphase+and+anaphase+and+facilitates+chromatid+motility.">The chromokinesin, KLP3A, dives mitotic spindle pole separation during prometaphase and anaphase and facilitates chromatid motility.</a> Mol Biol Cell. 15, 219-233 (2004).</li><li>Sharp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+coordination+of+three+mitotic+motors+in+Drosophila+embryos.">Functional coordination of three mitotic motors in Drosophila embryos.</a> Mol Biol Cell. 11, 241-253 (2000).</li><li>Sharp, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bipolar+kinesin,+KLP61F,+cross-links+microtubules+within+interpolar+microtubule+bundles+of+Drosophila+embryonic+mitotic+spindles.">The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles.</a> J Cell Biol. 144, 125-138 (1999).</li><li>Sharp, D. J., Rogers, G. C., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+dynein+is+required+for+poleward+chromosome+movement+during+mitosis+in+Drosophila+embryos.">Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos.</a> Nat Cell Biol. 2, 922-930 (2000).</li><li>Sharp, D. J., Yu, K. R., Sisson, J. C., Sullivan, W., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonistic+microtubule-sliding+motors+position+mitotic+centrosomes+in+Drosophila+early+embryos.">Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos.</a> Nat Cell Biol. 1, 51-54 (1999).</li><li>Waterman-Storer, C. M., Desai, A., Bulinski, J. C., Salmon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+speckle+microscopy,+a+method+to+visualize+the+dynamics+of+protein+assemblies+in+living+cells.">Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells.</a> Curr Biol. 8, 1227-1230 (1998).</li></ol>

Dieses Protokoll ist relativ einfach, aber jeder Schritt der Praxis erfordert, um sicherzustellen, dass die Embryonen nicht beschädigt werden. Sorgfältige Kontrolle Experimente brauchen immer zu tun, um sicherzustellen, dass zuverlässige Ergebnisse zu erhalten werden. Ein guter Weg, dies zu beginnen, ist durch Mikroinjektion Puffer oder Rhodamin Tubulin zu kontrollieren Embryonen, die GFP-Tubulin, um sicherzustellen, dass die Mitose in der Regel durchläuft alle Zyklen bei dem Embryo Oberfläche (Zyklen 10 bis 13). I...

microinjection techniques for studying mitosis in the drosophila melanogaster syncytial embryo

Dieses Protokoll beschreibt die Verwendung des Drosophila melanogaster-Syncytial-Embryo für das Studium der Mitose 1. Drosophila hat nützliche Genetik mit einem sequenzierten Genoms, und es kann leicht zu pflegen und manipuliert werden. Viele mitotischen Mutanten existieren, und transgenen Fliegen zum Ausdruck funktionaler fluoreszierend (zB GFP) - getaggt mitotischen Proteine ​​wurden und werden generiert. Gezielte Genexpression ist möglich mit dem GAL4/UAS System 2. Die Drosophila frühen Embryo führt mehrere Mitosen sehr schnell (Zellzyklus Dauer ≈ 10 min). Es ist gut für die Bildgebung der Mitose geeignet, da während der Zyklen 10-13, Kerne teilen sich rasch und synchron ohne einzugreifen Zytokinese an der Oberfläche des Embryos in einem einzigen Monoschicht knapp unterhalb der Hirnrinde. Diese sich schnell teilenden Kerne wahrscheinlich die gleichen mitotischen Maschinen wie andere Zellen, aber sie sind auf Geschwindigkeit optimiert; Kontrollpunkt wird allgemein angenommen, um nicht stringent, so dass das Studium der mitotischen Proteine, deren Fehlen würde Zellzyklusarrest in Zellen mit einem starken Checkpoint Ursache . Embryonen, die GFP-markierten Proteinen oder mikroinjiziert mit fluoreszenzmarkierten Proteine ​​können leicht abgebildet werden, um Live-Dynamik (Abb. 1) zu folgen. Darüber hinaus können Embryonen mit Funktions-blockierende Antikörper oder Inhibitoren von spezifischen Proteinen, um den Effekt des Verlustes oder der Störung ihrer Funktion 3-Studie mikroinjiziert werden. Diese Reagenzien können in den Embryo diffuse und erreichte viele Spindeln zu einem Gradienten der Konzentration des Inhibitors, was wiederum in einer Steigung von Mängeln vergleichbar mit einer allelischen Serie von Mutanten. Produzieren Im Idealfall, wenn das Zielprotein fluoreszierend markiert ist, kann die Steigung der Hemmung direkt dargestellt werden 4. Es wird davon ausgegangen, dass die stärksten Phänotyp vergleichbar mit der null-Phänotyp ist, obwohl es schwer ist, formal die Möglichkeit ausschließen, dass die Antikörper können dominanten Effekte in seltenen Fällen so strenge Kontrollen und eine vorsichtige Interpretation haben muss angewendet werden. Weiter entfernt von der Injektionsstelle ist Protein Funktion nur teilweise verloren damit andere Funktionen des Zielproteins sichtbar zu machen.

Watch this Scientific Journal Video about Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo at JoVE.com

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Dieses Protokoll beschreibt die Verwendung von Mikroinjektion und hochauflösende Bildgebung in der<em&gt; Drosophila melanogaster</em&gt; Syncytial Embryo Mitose studieren.

Mikroinjektion Techniken zur Untersuchung Mitose in der Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

microinjection-techniques-for-studying-mitosis-drosophila

Research

JoVE Journal

Biology

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

14.0K Aufrufe.  University of California, Davis. Dieses Protokoll beschreibt die Verwendung von Mikroinjektion und hochauflösende Bildgebung in der Drosophila melanogaster Syncytial Embryo Mitose studieren.

Serie: Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissection der Larven ZNS von Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Forschung

Biologie

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Hochauflösendes Video Tracking von Locomotion in Adult Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Elektrophysiologische Aufzeichnung in der Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

Überwachung Herzfunktion Larven Drosophila melanogaster Für physiologische Untersuchungen

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch-Clamp-und Perfusion Techniken zur Untersuchung von Ionenkanälen ausgedrückt in Xenopus Oozyten

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

Studieren mitotischen Checkpoint durch Veranschaulichung Dynamische Kinetochor Protein Verhalten und Chromosome Motion in Lebens-<em&gt; Drosophila</em&gt; Syncytial Embryonen

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Messung der Lebensdauer in<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Imaging Durch die Puppenhülle von<em&gt; Drosophila melanogaster</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

Verbesserte Northern Blot Detektion von kleinen RNA-Spezies in<em&gt; Drosophila melanogaster</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.