Ce travail a été soutenu par NIH NS042197 à MD. AJK a été soutenue par des subventions et des T32GM008719 F30NS068006.

1. Fabrication d&#39;aiguilles de microinjection <ol><li> Aiguilles de microinjection préfabriqués sont disponibles commercialement (par exemple. Femtotips d&#39;Eppendorf) et sont utiles si l&#39;on n&#39;est pas performant un grand nombre de micro-injections. Cependant, pour ceux qui souhaitent établir à long terme des capacités de micro-injection, une alternative est de produire des aiguilles de microinjection dans le laboratoire à l&#39;aide à paroi mince en verre borosilicate capillaires et un extracteur d&#39;aiguilles commerciales. Cela permet aussi la forme des aiguilles d&#39;être variée, qui peut être utile pour différents types cellulaires. </li><li> Avec l&#39;extracteur d&#39;aiguilles Narishige PC-10 microinjection, joindre tous les quatre poids et l&#39;utilisation d&#39;un programme en une seule étape traction (Étape 1 réglage) avec le réglage de chaleur relative à 58,0 (chauffage n ° 2). Veillez à placer l&#39;élément chauffant au centre de chaque capillaire afin que les deux aiguilles qui en résultent sont une longueur similaire. </li><li> Tirez plusieurs capillaires (environ 2 capillaires pour chaque protéine d&#39;intérêt). </li><li> Aiguilles Conserver dans un récipient tout en étant sûr de ne pas endommager la pointe de l&#39;aiguille. Des matériaux tels que mousse ou la Blu-Tack peut être utilisée dans des conteneurs à tenir les aiguilles de microinjection. </li></ol> 2. Préparation des mélanges de protéines pour injection <ol><li> Préparer un tampon microinjection 10x contenant 1 M de chlorure de potassium et de phosphate de potassium 0,1 M (KPI) tampon (mélange équimolaire de K 2 HPO 4 et KH 2 PO 4) à un pH de 7,4. Ce tampon peut être stocké à long terme à température ambiante. </li><li> Pour visualiser l&#39;injection, un colorant fluorescent comme la rhodamine-dextran doit être ajouté. Diluer tampon microinjection the10x dans l&#39;eau et dissoudre la rhodamine-dextran en poudre pour obtenir une solution tampon contenant microinjection 5x 20-40 mg / ml de rhodamine dextrane. </li><li> Conserver cette solution dans l&#39;obscurité à 4 ° C. Il devrait être suffisant pour jusqu&#39;à 100 préparations individuelles solution de protéines. D&#39;autres colorants, comme l&#39;isothiocyanate de fluorescéine dextrane peut se substituer. </li><li> Préparer le cytochrome c stocks en dissolvant purifiée cytochrome c dans l&#39;eau à une concentration de 20 mg / mL. Magasin de cytochrome c à -80 ° C pour stockage à long terme et d&#39;éviter les cycles de gel-dégel en stockant du cytochrome c dans les petites (~ 10 uL) aliquotes. </li><li> Préparer un mélange de protéines 10 uL pour injection par la combinaison de 2 pi de tampon 5x microinjection contenant rhodamine dextrane avec 3 d&#39;eau et 5 uL c uL cytochrome pour une concentration finale de 10 c mg / mL dans le cytochrome 1x tampon (100 mM KCl, 10 mM KP i, 4-8 mg / ml de rhodamine dextrane). </li></ol> 3. Micro-injection cytoplasmique du cytochrome C <ol><li> Juste avant la microinjection, centrifuger le mélange du cytochrome c et le tampon de la micro-injection à 16.000 g pendant 10 minutes à 4 ° C pour séparer les particules qui peuvent obstruer les aiguilles de microinjection. </li><li> Pendant la centrifugation, tournez sur la microinjecteur pour permettre à la pression d&#39;air à construire. </li><li> Placez un plat de cellules sur le centre de la platine du microscope et de mettre l&#39;accent sur les cellules. Afin de minimiser la quantité de temps que les cellules sont maintenues en dehors de l&#39;incubateur, les cellules sont normalement retournés à la couveuse dans les 30 min. </li><li> Pipeter 0,5 à 1 uL de la surface supérieure du mélange de protéines dans l&#39;extrémité émoussée de l&#39;capillaire. Attention à ne pas la pipette toutes les particules qui ont été centrifugés au fond du tube. En une minute, le mélange de protéines se distribuer à la pointe de l&#39;aiguille par capillarité. </li><li> Fixez l&#39;aiguille fermement à la porte capillaire du micromanipulateur et la position de l&#39;aiguille de sorte que sa pointe passe par la lumière transmise du microscope à environ un angle de 45 °. </li><li> Ajustez la position de l&#39;aiguille de sorte qu&#39;il est situé directement dans le centre du champ de vue. Pour centrer l&#39;aiguille, utilisez le micromanipulateur pour déplacer l&#39;aiguille tout en regardant dans l&#39;oculaire du microscope. L&#39;ombre aiguille doit être visible. Réglez l&#39;aiguille de telle sorte que son ombre ne se voit que dans une moitié du champ de vue, indiquant que la pointe de l&#39;aiguille est centrée au-dessus des cellules. </li><li> Réglez le microinjecteur au mode flux continu et régler la pression de travail à 20 - 100 hPa. Chaque aiguille peut exiger une pression différente de travail et la pression de travail sera probablement besoin d&#39;un ajustement lors de la procédure de micro-injection pour maintenir un flux constant. </li><li> Abaissez l&#39;aiguille en utilisant le bouton grossière à une position juste au-dessus des cellules. Pour ce faire, soulever le plan focal du microscope à une position juste au-dessus des cellules. Puis abaisser l&#39;aiguille vers les cellules en utilisant le bouton jusqu&#39;à ce que la grossière pointe de l&#39;aiguille est au point. </li><li> Recentrer l&#39;aiguille dans le champ de vision et augmenter le grossissement en changeant l&#39;objectif du microscope. </li><li> Abaisser lentement l&#39;aiguille à l&#39;aide du bouton de fines unti manipulateurl l&#39;aiguille est légèrement au-dessus du plan focal des cellules. </li><li> Vérifier l&#39;écoulement du mélange de protéines en regardant la fluorescence rouge de la rhodamine. Le mélange de protéines doit être sortie de l&#39;aiguille comme un mince jet constant. Les aiguilles doivent être remplacés et rechargé si l&#39;aiguille est compromise ou si le débit est beaucoup trop forte. Parfois, le bout du capillaire en verre est fermée et aucun débit est vu de l&#39;aiguille. Si cela se produit, remplacer l&#39;aiguille ou l&#39;abaissez au fond de la boîte de culture à la douceur de rupture de la pointe de l&#39;aiguille. </li><li> Placez le bout de l&#39;aiguille afin qu&#39;elle pointe vers une cellule à environ un angle de 45 °. Puis, avec un mouvement fluide, abaisser l&#39;aiguille tout en le déplaçant vers la cellule. Avec une deuxième motion en douceur, immédiatement inverser le sens de l&#39;aiguille pour l&#39;enlever de la cellule. </li><li> Une cellule de succès seront injectés souvent légèrement gonfler et peut être confirmé par la visualisation des rhodamine fluorescente rouge dans la cellule. Parfois, une cellule sera accidentellement injecté dans le noyau, ce qui sera visible. </li><li> Continuer à injecter des cellules en ajustant la platine du microscope jusqu&#39;à environ 50-100 cellules sont injectées. Lors du déplacement de la platine du microscope, assurez-vous de relever l&#39;aiguille de microinjection afin qu&#39;il efface le sommet des cellules. </li></ol> 4. Les résultats représentatifs: La micro-injection cytoplasmique de cytochrome c imite sa libération de mitochondries cours de l&#39;apoptose. Ainsi, comme attendu, les fibroblastes rapidement en apoptose après microinjection cytosolique de bovins du cytochrome c (figure 1A). Afin de s&#39;assurer que la procédure d&#39;injection elle seule n&#39;est pas responsable de la mort des cellules, l&#39;injection de levure cytochrome c sert un contrôle important, puisque la levure du cytochrome c est incapable d&#39;activer les caspases 12. Fait intéressant, post-mitotiques neurones sympathiques sont remarquablement résistants aux cytosolique du cytochrome c (Fig. 1B) 8,13. Notre laboratoire a identifié que la protéine XIAP caspases endogènes inhibiteur est un inhibiteur de la clé de l&#39;activation des caspases dans les neurones 14. Ainsi, pour les neurones de mourir après injection de cytochrome c, XIAP doit d&#39;abord devenir inactivé. Par exemple, la microinjection du cytochrome c dans XIAP - / - neurones sympathiques est suffisante pour permettre l&#39;activation des caspases et l&#39;apoptose dans ces cellules (Fig. 2). <img src="/files/ftp_upload/2773/2773fig1.jpg" alt="Figure 1"/> Figure 1. Micro-injection cytoplasmique du cytochrome c induit une mort rapide dans les fibroblastes, mais pas les neurones. A) de type sauvage FAE ou (B) 5 jours après la naissance de type sauvage neurones sympathiques ont été micro-injecté avec bovin cytochrome c (10 mg / mL) en collaboration avec la rhodamine-dextran pour marquer les cellules injectées. Les images montrent le même champ de cellules immédiatement après l&#39;injection (0 h), ou aux moments indiqués. Les flèches indiquent les cellules injectées. La barre d&#39;échelle, 20 um. <img src="/files/ftp_upload/2773/2773fig2.jpg" alt="Figure 2"/> Figure 2. XIAP déficientes neurones sont sensibles à la micro-injection cytoplasmique du cytochrome c. Postnatale jour 5 neurones sympathiques de souris knockout XIAP ont été micro-injecté avec bovin cytochrome c (10 mg / mL) en collaboration avec la rhodamine-dextran pour marquer les cellules injectées. Les images montrent le même champ de cellules immédiatement après l&#39;injection (0 h), ou 5 heures après le cytochrome c microinjection (5 h). La barre d&#39;échelle, 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. 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Toutes les procédures expérimentales sur les animaux ont été approuvés par le soin des animaux et du Comité institutionnel Utiliser à l&#39;Université de Caroline du Nord.

La micro-injection de cytochrome c directement dans le cytoplasme des cellules est un outil unique et puissant qui permet d&#39;études de la régulation post-mitochondriale de l&#39;apoptose. Fait important, cette technique permet l&#39;activation directe de l&#39;apoptose aval de la mitochondrie, sans l&#39;utilisation d&#39;agents qui causent des dommages cellulaires ou mitochondriales. Bien que ce protocole a porté sur la microinjection du cytochrome c pour les études...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> DM IRE2 microscope inversé </td><td> Leica </td><td></td><td></td></tr><tr><td> PC-10 Extracteur d&#39;aiguilles microinjection </td><td> Narishige </td><td></td><td></td></tr><tr><td> Adjum-202 Micromanipulateur </td><td> Narishige </td><td></td><td></td></tr><tr><td> FemtoJet microinjecteur </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> Paroi mince Boroscilicate capillaire en verre avec Microfilament </td><td> Systèmes AM </td><td> 615000 </td><td> 4 pouces de longueur, 1,00 mm de diamètre extérieur, 0,75 mm de diamètre interne </td></tr><tr><td> Rhodamine B isothiocyanate-Dextran </td><td> Sigma-Aldrich </td><td> R9379 </td><td> Poids moléculaire moyen 70 000 Da ~ </td></tr><tr><td> Bovine Cytochrome protéine C </td><td> Sigma-Aldrich </td><td> C3131 </td><td></td></tr></tbody></table>

activation of apoptosis by cytoplasmic microinjection of cytochrome c

L&#39;apoptose ou mort cellulaire programmée, est une voie conservées et très réglementé par lequel les cellules meurent 1. L&#39;apoptose peut être déclenchée quand les cellules rencontrent une large gamme de contraintes cytotoxiques. Ces insultes provoquent des cascades de signalisation qui finissent par provoquer la libération du cytochrome c de l&#39;espace intermembranaire mitochondrial vers le cytoplasme 2. La libération du cytochrome c de la mitochondrie est un événement clé qui déclenche l&#39;activation rapide des caspases, les protéases cellulaires clés qui finalement exécuter la mort cellulaire 3-4. La voie de l&#39;apoptose est régulée dans les points amont et aval de la libération du cytochrome c à partir de 5 mitochondries. Afin d&#39;étudier la régulation post-mitochondrial de l&#39;activation des caspases, de nombreux chercheurs se sont tournés vers la micro-injection cytoplasmique directe de holocytochrome c (hème-jointe) de protéines dans les cellules 6-9. Cytochrome c est normalement localisée dans les mitochondries, où l&#39;attachement d&#39;un groupe hème est nécessaire pour lui permettre d&#39;activer l&#39;apoptose 10-11. Par conséquent, pour activer directement les caspases, il est nécessaire d&#39;injecter la protéine holocytochrome c au lieu de son ADNc, parce que l&#39;expression du cytochrome c de constructions d&#39;ADNc se traduira dans le ciblage mitochondrial et l&#39;attachement hème, il sera séquestré par les caspases cytosolique. Ainsi, la micro-injection directe cytosolique de la protéine purifiée hème-joint le cytochrome c est un outil utile pour imiter cytochrome c mitochondrial libération et l&#39;apoptose sans l&#39;utilisation d&#39;agressions toxiques qui provoquent des dommages cellulaires et mitochondriales. Dans cet article, nous décrivons une méthode pour la microinjection du cytochrome c de protéines dans les cellules, en utilisant la souris fibroblastes embryonnaires (MEF) et primaires neurones sympathiques comme exemples. Alors que ce protocole met l&#39;accent sur ​​l&#39;injection de cytochrome c pour les enquêtes de l&#39;apoptose, les techniques présentées ici peuvent également être facilement adapté à la micro-injection d&#39;autres protéines d&#39;intérêt.

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

Dans ce protocole, nous décrivons la micro-injection directe cytoplasmique du cytochrome<em&gt; C</emProtéines&gt; dans des fibroblastes primaires et des neurones sympathiques. Cette technique permet l&#39;introduction du cytochrome<em&gt; C</emProtéines&gt; dans le cytoplasme des cellules et imite la libération du cytochrome<em&gt; C</em&gt; À partir de mitochondries, ce qui se produit lors de l&#39;apoptose.

L&#39;activation de l&#39;apoptose par micro-injection cytoplasmique du cytochrome 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 vues.  University of North Carolina. Dans ce protocole, nous décrivons la micro-injection directe cytoplasmique du cytochrome C dans des fibroblastes primaires et des neurones sympathiques. Cette technique permet l'introduction du cytochrome C dans le cytoplasme des cellules et imite la libération du cytochrome C À partir de mitochondries, ce qui se produit lors de l'apoptose.

Vidéo: 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: présentation des antigènes glycolipides par CD1d et le potentiel thérapeutique de l&#39;activation des cellules NKT

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

Recherche

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.

Génération de Stable elegans transgéniques C. Utilisation de microinjection

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.

Microinjection d&#39;ovocytes de Xenopus laevis

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.

Génération de souris transgéniques C. elegans Par transformation biolistique

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.

Analyse de la cinétique ARNm exportation nucléaire dans les cellules de mammifères par micro-injection

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.

Analyse de la fonction des petites GTPases par micro-injection de plasmides dans les cellules épithéliales polarisées

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.

Visualisation de la fonction respiratoire mitochondriale utilisant Cytochrome C Oxydase / succinate déshydrogénase (COX / SDH) Double-histochimie étiquetage

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.

Synthèse d&#39;un<em&gt; In vivo</em&gt; Sonde apoptose détectable par IRM

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.

Une seule molécule d&#39;imagerie du règlement Gene<em&gt; In vivo</em&gt; Utilisation de l&#39;activation par clivage cotraductionnelle (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.

Analyse de l&#39;apoptose dans embryons de poissons zèbres par Whole montage immunofluorescence pour détecter caspase 3

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