Diese Arbeit wurde vom NIH NS042197 zu MD unterstützt. AJK wurde durch Zuschüsse T32GM008719 und F30NS068006 unterstützt.

1. Produktion von Mikroinjektion Needles <ol><li> Vorgefertigte Mikroinjektion Nadeln sind im Handel erhältlich (z. B.. Femtotips von Eppendorf) und sind nützlich, wenn man nicht die Durchführung einer großen Anzahl von microinjections. Doch für diejenigen, die langfristigen Möglichkeiten für Mikroinjektion aufbauen wollen, ist eine Alternative zur Mikroinjektion Nadeln im Labor mit dünnwandigen Borosilikatglas Kapillaren und eine kommerzielle Nadel puller produzieren. Dies ermöglicht auch die Form von Nadeln variiert werden, was nützlich sein kann für verschiedene Zelltypen. </li><li> Mit dem Narishige PC-10 Mikroinjektionsnadel Puller, legen alle vier Gewichte und benutzen Sie eine Ein-Schritt-Zug-Programms (Schritt 1 Einstellung) mit der relativen Wärme-Einstellung bei 58,0 (No.2 Heizung). Achten Sie darauf, das Heizelement in der Mitte jeder Kapillare so Platz, dass die beiden entstehenden Nadeln eine ähnliche Länge haben. </li><li> Ziehen Sie mehrere Kapillaren (ca. 2 Kapillaren für jedes Protein von Interesse). </li><li> Shop Nadeln in einem Container, während sie sicher nicht an der Nadelspitze Schäden. Materialien wie Schaumstoff oder Blu-Tack können in Containern eingesetzt werden, um die Mikroinjektion Nadeln halten. </li></ol> 2. Herstellung von Protein-Mischungen für die Injektion <ol><li> Bereiten Sie eine 10-Mikroinjektion Puffer mit 1 M Kaliumchlorid und 0,1 M Kaliumphosphatpuffer (KPI)-Puffer (äquimolare Mischung aus K 2 HPO 4 und KH 2 PO 4) bei einem pH von 7,4. Dieser Puffer kann langfristig aufbewahrt bei Raumtemperatur. </li><li> Zur Visualisierung der Injektion muss ein fluoreszierender Farbstoff wie Rhodamin-Dextran hinzugefügt werden. Verdünnen the10x Mikroinjektion Puffer in Wasser auflösen und Rhodamin-Dextran Pulver zu einer 5x Mikroinjektion Pufferlösung mit 20-40 mg / mL Rhodamin Dextran zu machen. </li><li> Bewahren Sie diese Lösung im Dunkeln bei 4 ° C. Es sollte ausreichen, für bis zu 100 einzelnen Protein-Lösung Vorbereitungen. Andere Farbstoffe, wie Fluoresceinisothiocyanat-Dextran ersetzen können. </li><li> Bereiten Cytochrom c Aktien durch Auflösen von gereinigtem Cytochrom c in Wasser zu einer Konzentration von 20 mg / mL. Shop Cytochrom c bei -80 ° C für die langfristige Lagerung und zur Vermeidung von Frost-Tau-Zyklen durch die Speicherung Cytochrom c in klein (~ 10 l) Aliquots. </li><li> Bereiten Sie eine 10 l Protein-Mischung für die Injektion durch die Kombination von 2 ul 5x Mikroinjektion Puffer mit Rhodamin-Dextran mit 3 ul Wasser und 5 ul Cytochrom c für eine endgültige Konzentration von 10 mg / mL Cytochrom c in 1x Puffer (100 mM KCl, 10 mM KP i, 4-8 mg / mL Rhodamin Dextran). </li></ol> 3. Zytoplasmatische Mikroinjektion von Cytochrom c <ol><li> Kurz vor der Mikroinjektion, Zentrifuge die Mischung von Cytochrom c und Mikroinjektion Puffer bei 16.000 g für 10 Minuten bei 4 ° C zu jedem Partikel, die Mikroinjektion Nadeln verstopfen können zu trennen. </li><li> Während der Zentrifugation auf die Mikroinjektor wiederum zu ermöglichen Luftdruck zu bauen. </li><li> Legen Sie ein Gericht von Zellen in der Mitte des Mikroskoptisches und den Fokus auf die Zellen. Zur Minimierung der Menge an Zeit, dass die Zellen außerhalb des Inkubators gehalten werden, werden die Zellen in der Regel in den Inkubator innerhalb von 30 min zurück. </li><li> Pipette 0,5-1 ul von der Oberfläche des Proteins Mischung in das stumpfe Ende der Kapillare. Seien Sie vorsichtig, um nicht Pipette alle Partikel, die an der Unterseite des Rohres wurden zentrifugiert. Innerhalb einer Minute wird das Proteingemisch auf die Nadelspitze durch Kapillarwirkung zu verteilen. </li><li> Befestigen Sie die Nadel fest mit der Kapillare Inhaber der Mikromanipulator und die Position der Nadel so dass seine Spitze durch das übertragene Licht des Mikroskops verläuft bei ca. 45 °-Winkel. </li><li> Passen Sie die Position der Nadelspitze, so dass es direkt in der Mitte des Sichtfeldes liegt. Zur Zentrierung der Nadel, mit der Mikromanipulator, um die Nadel zu bewegen, während Sie durch das Mikroskop-Okular. Die Nadel Schatten sichtbar sein soll. Stellen Sie die Nadel so, dass ihr Schatten ist nur in einer Hälfte des Gesichtsfeldes zu sehen, was darauf hinweist, dass die Nadelspitze über den Zellen zentriert ist. </li><li> Stellen Sie die Mikroinjektor der Continuous Flow-Modus und stellen Sie den Betriebsdruck auf 20 bis 100 hPa. Jede Nadel kann eine andere Betriebsdruck und der Betriebsdruck wird wahrscheinlich angepasst werden müssen während der Mikroinjektion Verfahren, um einen stetigen Fluss zu halten. </li><li> Senken Sie die Nadel mit dem groben Drehknopf um eine Position oberhalb der Zellen. Um dies zu tun, heben Sie die Fokusebene des Mikroskops in eine Position oberhalb der Zellen. Dann senken Sie die Nadel auf die Zellen unter Verwendung des groben Regler, bis die Nadelspitze im Fokus ist. </li><li> Re-Center die Nadelspitze in das Sichtfeld und die Vergrößerung zu erhöhen, indem Sie die Mikroskop-Objektiv. </li><li> Langsam senken Sie die Nadel mit dem feinen Manipulator Knopf until die Nadel knapp über der Brennebene der Zellen. </li><li> Überprüfen Sie den Ablauf der Protein-Mischung, indem man die rote Fluoreszenz des Rhodamin. Das Protein Mischung sollte Verlassen der Nadel als eine dünne, konstanten Strom. Needles sollte ersetzt werden und wieder geladen werden, wenn die Nadel gefährdet ist oder wenn die Strömung ist viel zu stark. Manchmal ist die Spitze der Glaskapillare geschlossen und keine Strömung wird von der Nadel zu sehen. Wenn dies der Fall, ersetzen Sie die Nadel oder sorgfältig niedrigeren es auf den Boden der Kulturschale zu sanft Bruch der Spitze der Nadel. </li><li> Position der Nadelspitze, so dass es zu einer Zelle verweist auf ca. 45 °-Winkel. Dann mit einer geschmeidigen Bewegung, senken Sie die Nadel, während Sie sie in Richtung der Zelle. Mit einer zweiten glatten Bewegung, sofort rückwärts Richtung der Nadel, um sie aus der Zelle zu entfernen. </li><li> Ein erfolgreich injizierten Zellen wird oft leicht anschwellen und kann durch die Visualisierung der rot fluoreszierende Rhodamin innerhalb der Zelle zu bestätigen. Gelegentlich wird eine Zelle versehentlich in den Zellkern, die sichtbar sein wird injiziert werden. </li><li> Fahren Sie mit Zellen, die durch die Anpassung der Mikroskoptisch bis etwa 50-100 Zellen injiziert werden zu injizieren. Beim Bewegen des Mikroskops, müssen Sie die Mikroinjektionsnadel erhöhen, so dass es Anfang der Zellen löscht. </li></ol> 4. Repräsentative Ergebnisse: Die cytoplasmatische Mikroinjektion von Cytochrom c ahmt seine Freisetzung aus Mitochondrien während der Apoptose. So, wie erwartet, Fibroblasten schnell Apoptose auf zytosolische Mikroinjektion von Rindern Cytochrom c (Abb. 1A). Um sicherzustellen, dass die Injektion allein nicht verantwortlich für den Zelltod, dient Injektion von Hefe Cytochrom c als eine wichtige Kontrolle, da Hefe Cytochrom c ist unfähig, die Aktivierung Caspasen 12. Interessanterweise sind post-mitotischen sympathischen Neuronen bemerkenswert widerstandsfähig gegenüber cytosolischen Cytochrom c (Abb. 1B) 8,13. Unser Labor hat festgestellt, dass die endogene Caspase-Inhibitor XIAP eine Schlüsselrolle Inhibitor der Caspase-Aktivierung in Neuronen 14 ist. So Neuronen nach Cytochrom c Injektion sterben müssen XIAP werden erst inaktiviert. Zum Beispiel, Mikroinjektion von Cytochrom c in XIAP - wird sympathischen Neuronen ausreichend, um Caspase-Aktivierung und Apoptose in diesen Zellen (Abb. 2) ermöglichen - /. <img src="/files/ftp_upload/2773/2773fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Zytoplasmatische Mikroinjektion von Cytochrom c induziert einen schnellen Tod in Fibroblasten, aber nicht Neuronen. A) Wildtyp-MEFs oder (B) postnatalen Tag 5 Wildtyp-sympathischen Neuronen wurden mit Rinder-Cytochrom c (10 mg / ml) zusammen mit Rhodamin-Dextran injizierten Zellen markieren mikroinjiziert. Bilder zeigen den gleichen Bereich der Zellen unmittelbar nach der Injektion (0 h), oder zu den angegebenen Zeiten. Die Pfeile zeigen die injizierten Zellen. Scale-bar, 20 um. <img src="/files/ftp_upload/2773/2773fig2.jpg" alt="Abbildung 2"/> Abbildung 2. XIAP-defizienten Neuronen anfälliger für zytoplasmatische Cytochrom c Mikroinjektion. Postnatale Tag 5 sympathischen Neuronen von XIAP-Knockout-Mäusen wurden mit Rinder-Cytochrom c (10 mg / ml) zusammen mit Rhodamin-Dextran injizierten Zellen markieren mikroinjiziert. Bilder zeigen den gleichen Bereich der Zellen unmittelbar nach der Injektion (0 h) oder 5 Stunden nach der Cytochrom-c-Mikroinjektion (5 Std.). Scale-bar, 20 um.

<ol>
	<li>Danial, N. N., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death:+critical+control+points.">Cell death: critical control points.</a> Cell. 116, 205-219 (2004).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+role+of+mitochondria+in+apoptosis.">The expanding role of mitochondria in apoptosis.</a> Genes Dev. 15, 2922-2933 (2001).</li><li>Hengartner, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemistry+of+apoptosis.">The biochemistry of apoptosis.</a> Nature. 407, 770-776 (2000).</li><li>Fuentes-Prior, P., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+structures+that+shape+caspase+activity,+specificity,+activation+and+inhibition.">The protein structures that shape caspase activity, specificity, activation and inhibition.</a> Biochem. J. 384, 201-232 (2004).</li><li>Tait, S. W., Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+cell+death:+outer+membrane+permeabilization+and+beyond.">Mitochondria and cell death: outer membrane permeabilization and beyond.</a> Nat Rev Mol Cell Biol. 11, 621-632 (2010).</li><li>Brustugun, O. T., Fladmark, K. E., Doskeland, S. O., Orrenius, S., Zhivotovsky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+induced+by+microinjection+of+cytochrome+c+is+caspase-dependent+and+is+inhibited+by+Bcl-2.">Apoptosis induced by microinjection of cytochrome c is caspase-dependent and is inhibited by Bcl-2.</a> Cell Death Differ. 5, 660-668 (1998).</li><li>Li, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+induction+of+apoptosis+by+microinjection+of+cytochrome+c.+Bcl-XL+has+activity+independent+of+cytochrome+c+release.">Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-XL has activity independent of cytochrome c release.</a> J. Biol. Chem. 272, 30299-30305 (1997).</li><li>Deshmukh, M., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+novel+event+during+neuronal+death:+development+of+competence-to-die+in+response+to+cytoplasmic+cytochrome+c.">Evidence of a novel event during neuronal death: development of competence-to-die in response to cytoplasmic cytochrome c.</a> Neuron. 21, 695-705 (1998).</li><li>Vaughn, A. E., Deshmukh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+metabolism+inhibits+apoptosis+in+neurons+and+cancer+cells+by+redox+inactivation+of+cytochrome+c.">Glucose metabolism inhibits apoptosis in neurons and cancer cells by redox inactivation of cytochrome c.</a> Nat. Cell Biol. 10, 1477-1483 (2008).</li><li>Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+apoptosis+by+Bcl-2:+release+of+cytochrome+c+from+mitochondria+blocked.">Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.</a> Science. 275, 1129-1132 (1997).</li><li>Gonzales, D. H., Neupert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+mitochondrial+c-type+cytochromes.">Biogenesis of mitochondrial c-type cytochromes.</a> J Bioenerg Biomembr. 22, 753-768 (1990).</li><li>Ellerby, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+cell-free+system+of+neuronal+apoptosis+-+comparison+of+premitochondrial,+mitochondrial,+and+postmitochondrial+phases.">Establishment of a cell-free system of neuronal apoptosis - comparison of premitochondrial, mitochondrial, and postmitochondrial phases.</a> J. Neurosci. 17, 6165-6178 (1997).</li><li>Neame, S. J., Rubin, L. L., Philpott, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+cytochrome+c+activity+within+intact+neurons+inhibits+apoptosis.">Blocking cytochrome c activity within intact neurons inhibits apoptosis.</a> J. Cell Biol. 142, 1583-1593 (1998).</li><li>Potts, P. R., Singh, S., Knezek, M., Thompson, C. B., Deshmukh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+function+of+endogenous+XIAP+in+regulating+caspase+activation+during+sympathetic+neuronal+apoptosis.">Critical function of endogenous XIAP in regulating caspase activation during sympathetic neuronal apoptosis.</a> J. Cell Biol. 163, 789-799 (2003).</li><li>Estus, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+gene+expression+in+neurons+during+programmed+cell+death:+identification+of+c-jun+as+necessary+for+neuronal+apoptosis.">Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis.</a> J. Cell Biol. 127, 1717-1727 (1994).</li></ol>

Alle Experimente an Tieren wurden von der Institutional Animal Care und Verwenden Ausschuss an der University of North Carolina genehmigt.

Die Mikroinjektion von Cytochrom c direkt in das Zytoplasma der Zellen ist ein einzigartiges und leistungsfähiges Werkzeug, das für die Untersuchung der post-mitochondrialen Regulation der Apoptose ermöglicht. Wichtig ist, ermöglicht diese Technik für die direkte Aktivierung der Apoptose stromabwärts von Mitochondrien ohne den Einsatz von Mitteln, die zellulären oder mitochondriale Schäden verursachen. Während dieses Protokoll auf die Mikroinjektion von Cytochrom c...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> DM IRE2 Inverses Mikroskop </td><td> Leica </td><td></td><td></td></tr><tr><td> PC-10 Mikroinjektionsnadel Puller </td><td> Narishige </td><td></td><td></td></tr><tr><td> MWO-202 Mikromanipulator </td><td> Narishige </td><td></td><td></td></tr><tr><td> FemtoJet Mikroinjektor </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> Dünnwandige Boroscilicate Kapillar-Glas mit Microfilament </td><td> AM-Systeme </td><td> 615000 </td><td> 4 inch Länge, 1,00 mm Außendurchmesser, 0,75 mm Innendurchmesser </td></tr><tr><td> Rhodamin B-Isothiocyanat-Dextran </td><td> Sigma-Aldrich </td><td> R9379 </td><td> Durchschnittliches Molekulargewicht ~ 70.000 Da </td></tr><tr><td> Bovine Cytochrom c Protein </td><td> Sigma-Aldrich </td><td> C3131 </td><td></td></tr></tbody></table>

activation of apoptosis by cytoplasmic microinjection of cytochrome c

Apoptose oder der programmierte Zelltod, ist eine konservierte und stark regulierten Weg durch die Zellen sterben 1. Apoptose ausgelöst, wenn die Zellen Begegnung eine breite Palette von zytotoxischen betont werden. Diese Beleidigungen initiieren Signalkaskaden, die letztlich zur Freisetzung von Cytochrom c aus den Mitochondrien Intermembranraum in das Cytoplasma 2. Die Freisetzung von Cytochrom c aus den Mitochondrien ist ein wichtiges Ereignis, dass die schnelle Aktivierung von Caspasen, die wichtigsten zellulären Proteasen, die letztlich zum Zelltod führen 3-4 auslöst. Der Weg der Apoptose wird an den Punkten vor-und nachgelagerten von Cytochrom c Freisetzung aus Mitochondrien 5 geregelt. Um die post-mitochondrialen Regulation der Caspase-Aktivierung Studie haben viele Forscher den direkten cytoplasmatischen Mikroinjektion von Holocytochrom c (Häm-attached)-Protein in Zellen 6-9 gedreht. Cytochrom c ist in der Regel in die Mitochondrien, wo Anbringung eines Häm-Gruppe ist notwendig, damit es zur Apoptose 10-11 aktivieren lokalisiert. Deshalb, um direkt zu aktivieren Caspasen, ist es notwendig, die Holocytochrom c Protein anstelle des cDNA injizieren, denn während die Expression von Cytochrom c aus cDNA-Konstrukte in der mitochondrialen Targeting-und Häm-Anlage führen wird, ist es von der zytosolischen Caspasen beschlagnahmt werden. So ist die direkte cytosolische Mikroinjektion von gereinigtem Häm-attached Cytochrom c-Protein ein nützliches Instrument zur mitochondrialen Cytochrom c Freisetzung und Apoptose, ohne die Verwendung von giftigen Beleidigungen, die zellulären und mitochondrialen Schäden verursachen imitieren. In diesem Artikel beschreiben wir eine Methode für die Mikroinjektion von Cytochrom c-Proteins in Zellen mit embryonalen Maus-Fibroblasten (MEFs) und der primären sympathischen Neuronen als Beispiele. Während dieses Protokoll konzentriert sich auf die Injektion von Cytochrom c für Untersuchungen der Apoptose, können die hier gezeigten Techniken auch leicht für die Mikroinjektion der andere Proteine ​​von Interesse angepasst werden.

Watch this Scientific Journal Video about Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c at JoVE.com

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In diesem Protokoll, beschreiben wir die direkte zytoplasmatische Mikroinjektion von Cytochrom<em&gt; C</em&gt; Protein in Fibroblasten und primäre sympathischen Neuronen. Diese Technik ermöglicht die Einführung von Cytochrom<em&gt; C</em&gt; Protein in das Zytoplasma der Zellen und ahmt die Freisetzung von Cytochrom<em&gt; C</em&gt; Aus den Mitochondrien, die auftritt, während der Apoptose.

Die Aktivierung der Apoptose durch cytoplasmatische Mikroinjektion von Cytochrom C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

activation-of-apoptosis-by-cytoplasmic-microinjection-of-cytochrome-c

Research

JoVE Journal

Biology

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

17.3K Aufrufe.  University of North Carolina. In diesem Protokoll, beschreiben wir die direkte zytoplasmatische Mikroinjektion von Cytochrom C Protein in Fibroblasten und primäre sympathischen Neuronen. Diese Technik ermöglicht die Einführung von Cytochrom C Protein in das Zytoplasma der Zellen und ahmt die Freisetzung von Cytochrom C Aus den Mitochondrien, die auftritt, während der Apoptose.

Serie: Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques.   In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.  Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells.  The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells.  Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy.  Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmunity, clearance of infection, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.

Interview: Glycolipid Antigen-Präsentation durch CD1d und das therapeutische Potenzial von NKT Zellen-Aktivierung

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious ...

Forschung

Biologie

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Generierung von stabilen transgenen C. elegans mit Mikroinjektion

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Mikroinjektion von Xenopus laevis Oozyten

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Erzeugung von transgenen C. elegans Nach Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Die Analyse der mRNA Nuclear Export Kinetics in Säugerzellen durch Mikroinjektion

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Die Analyse der Funktion von kleinen GTPasen durch Mikroinjektion von Plasmiden in polarisierten Epithelzellen

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Visualisierung der mitochondrialen Atmungskette Funktion mit Cytochrom C Oxidase / Succinat-Dehydrogenase (COX / SDH) Double-Kennzeichnung Histochemie

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

Synthese eines<em&gt; In vivo</em&gt; MRT-Probe nachweisbar Apoptose

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Einzel-Molekül-Imaging der Genregulation<em&gt; In vivo</em&gt; Verwenden cotranslationalen Aktivierung durch Spaltung (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Die Analyse der Apoptose in Zebrafischembryonen von Whole-mount Immunfluoreszenz an aktivierte Caspase 3 Detect

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...