Questo lavoro è stato sostenuto da NIH concedere NS042197 a MD. AJK è stato supportato da sovvenzioni T32GM008719 e F30NS068006.

1. Produzione di aghi Microiniezione <ol><li> Prefabbricati aghi microiniezione Sono disponibili in commercio (ad es. Femtotips da Eppendorf) e sono utili se non si sta eseguendo un gran numero di microiniezioni. Tuttavia, per coloro che desiderano stabilirsi a lungo termine le capacità per microinjecting, un&#39;alternativa è quella di produrre aghi microiniezione in laboratorio utilizzando sottile parete in vetro borosilicato capillari e un estrattore ago commerciale. Questo permette anche la forma degli aghi per essere variato, che può essere utile per diversi tipi di cellule. </li><li> Con il PC-10 Microiniezione Puller Ago Narishige, allegare tutti e quattro i pesi e utilizzare un one-step programma tirando (Fase 1 impostazione) con l&#39;impostazione di calore rispetto a 58,0 (riscaldamento n.2). Assicurarsi di posizionare l&#39;elemento riscaldante al centro di ogni capillare in modo che i due aghi che costituiscano una lunghezza simile. </li><li> Tirare diversi capillari (circa 2 capillari per ogni proteina di interesse). </li><li> Aghi Conservare in un contenitore pur essendo sicuri di non danneggiare la punta dell&#39;ago. Materiali come schiuma o Blu-Tack può essere utilizzato in contenitori per contenere aghi microiniezione. </li></ol> 2. Preparazione di miscele di proteine ​​per iniezione <ol><li> Preparare un buffer 10x microiniezione contenente 1 M di cloruro di potassio e fosfato di potassio 0,1 M (KPI) buffer (miscela equimolare di K 2 HPO 4 e KH 2 PO 4) ad un pH di 7,4. Questo buffer può essere conservato a lungo termine a temperatura ambiente. </li><li> Per visualizzare l&#39;iniezione, un colorante fluorescente come rodamina-destrano deve essere aggiunto. Diluire il tampone the10x microiniezione in acqua e sciogliere rodamina-destrano in polvere per fare una soluzione tampone 5x microiniezione contenente 20-40 mg / mL rodamina destrano. </li><li> Conservare questa soluzione al buio a 4 ° C. Dovrebbe essere sufficiente per un massimo di 100 preparazioni individuali soluzione proteica. Altri coloranti, come la fluoresceina isotiocianato-destrano può sostituire. </li><li> Preparare citocromo c scorte sciogliendo purificato citocromo c in acqua ad una concentrazione di 20 mg / mL. Conservare citocromo c a -80 ° C per conservazione a lungo termine ed evitare cicli gelo-disgelo, memorizzando citocromo c in piccole (~ 10 mL) aliquote. </li><li> Preparare una miscela di 10 microlitri di proteine ​​per l&#39;iniezione, combinando 2 il buffer microlitri microiniezione 5x contenente rodamina-destrano con 3 microlitri di acqua e 5 microlitri citocromo c per una concentrazione finale di 10 mg / ml citocromo c in 1x tampone (100 mM KCl, 10 mM KP i, 4-8 mg / mL rodamina destrano). </li></ol> 3. Microiniezione citoplasmatica della citocromo c <ol><li> Appena prima di microiniezione, centrifuga la miscela di citocromo c ed tampone microiniezione a 16.000 g per 10 minuti a 4 ° C per separare le particelle che potrebbero intasare gli aghi microiniezione. </li><li> Durante la centrifugazione, accendere il microinjector per consentire la pressione dell&#39;aria a costruire. </li><li> Posizionare un piatto di cellule al centro del palco microscopio e impostare lo stato attivo sulle cellule. Per ridurre al minimo la quantità di tempo che sono conservate le cellule al di fuori dell&#39;incubatore, le cellule sono di solito restituiti al incubatore entro 30 min. </li><li> Pipettare 0,5-1 microlitri dalla superficie superiore della miscela proteica nelle estremità piatta del capillare. Fare attenzione a non pipetta le particelle che sono stati centrifugati al fondo della provetta. Nel giro di un minuto, la miscela proteica distribuirà alla punta dell&#39;ago attraverso l&#39;azione capillare. </li><li> Applicare l&#39;ago saldamente al titolare capillare del micromanipolatore e la posizione della lancetta in modo che la punta passa attraverso la luce trasmessa del microscopio a circa 45 °. </li><li> Regolare la posizione della punta dell&#39;ago in modo che si trova nel centro del campo visivo. Per centrare l&#39;ago, utilizzare il micromanipolatore per spostare l&#39;ago mentre guardando attraverso l&#39;oculare microscopio. L&#39;ombra ago deve essere visibile. Regolare l&#39;ago in modo che la sua ombra è visto solo in una metà del campo visivo, indicando che la punta dell&#39;ago è centrata sopra le cellule. </li><li> Impostare il microinjector alla modalità a flusso continuo e regolare la pressione di lavoro a 20 - 100 hPa. Ogni ago può richiedere una diversa pressione di lavoro, e la pressione di lavoro sarà probabilmente necessario aggiustamento durante la procedura di microiniezione per mantenere un flusso costante. </li><li> Abbassare l&#39;ago con la manopola grossolana a una posizione appena sopra le cellule. Per fare questo, aumentare il piano focale del microscopio di una posizione appena sopra le cellule. Quindi abbassare l&#39;ago verso le cellule usando la manopola grossolana fino a quando la punta dell&#39;ago è a fuoco. </li><li> Ricentrare la punta dell&#39;ago all&#39;interno del campo visivo e aumentare l&#39;ingrandimento cambiando l&#39;obiettivo microscopio. </li><li> Abbassare lentamente l&#39;ago con la multa unti manopola manipolatorel l&#39;ago è leggermente al di sopra del piano focale delle cellule. </li><li> Controllare il flusso della miscela proteica guardando la fluorescenza rossa della rodamina. La miscela di proteine ​​dovrebbe essere uscire l&#39;ago come un sottile flusso costante. Gli aghi devono essere sostituiti e ri-caricato se l&#39;ago è compromessa o se il flusso è troppo forte. A volte, la punta di un capillare di vetro è chiuso e nessun flusso è visto dall&#39;ago. In questo caso, sostituire l&#39;ago o accuratamente inferiore al fondo del piatto cultura di rottura con delicatezza la punta dell&#39;ago. </li><li> Posizionare la punta dell&#39;ago in modo che sia rivolta verso una cella di circa 45 °. Poi con un movimento fluido, abbassare l&#39;ago durante lo spostamento verso la cella. Con una seconda mozione liscio, immediatamente invertire la direzione dell&#39;ago per rimuoverlo dalla cellula. </li><li> Una cellula con successo iniettato spesso un po &#39;gonfia e può essere confermata dalla visualizzazione della rodamina rosso fluorescente all&#39;interno della cellula. Occasionalmente, una cella sarà accidentalmente iniettato nel nucleo, che sarà visibile. </li><li> Continuano a iniettare le cellule regolando la fase microscopio fino a circa 50-100 cellule vengono iniettate. Quando si sposta la fase di microscopio, assicurarsi di sollevare l&#39;ago microiniezione in modo che cancella la parte superiore delle cellule. </li></ol> 4. Rappresentante dei risultati: La microiniezione citoplasmatico del citocromo c imita il suo rilascio dai mitocondri durante l&#39;apoptosi. Così, come previsto, i fibroblasti rapidamente apoptosi su microiniezione citosolico di bovini citocromo c (Fig. 1A). Per garantire che la procedura di iniezione da sola non è responsabile per la morte cellulare, l&#39;iniezione di lievito citocromo c serve come un controllo importante, dal momento lievito citocromo c è in grado di attivare caspasi 12. È interessante notare che post-mitotico neuroni simpatici sono molto resistenti a citosolico citocromo c (Fig. 1B) 8,13. Il nostro laboratorio ha identificato che la XIAP inibitore endogeno della caspasi è un inibitore chiave di attivazione delle caspasi nei neuroni 14. Così, per i neuroni a morire dopo l&#39;iniezione del citocromo c, XIAP deve prima diventare inattivato. Per esempio, la microiniezione di citocromo c in XIAP - / - neuroni simpatici è sufficiente per consentire l&#39;attivazione delle caspasi e apoptosi in queste cellule (Fig. 2). <img src="/files/ftp_upload/2773/2773fig1.jpg" alt="Figura 1"/> Figura 1. Microiniezione citoplasmatico del citocromo c induce la morte rapida nei fibroblasti, ma non i neuroni. A) wild-type o MEF (B) giorno dopo la nascita 5 neuroni simpatici wild-type sono state microiniettati bovina con citocromo c (10 mg / mL) insieme con rodamina-destrano per marcare le cellule iniettate. Le immagini mostrano lo stesso campo di cellule immediatamente dopo l&#39;iniezione (0 ore), oppure secondo gli orari indicati. Le frecce indicano le cellule iniettate. Scala grafica, 20 micron. <img src="/files/ftp_upload/2773/2773fig2.jpg" alt="Figura 2"/> Figura 2. XIAP deficit di neuroni sono suscettibili di citoplasma del citocromo c microiniezione. Postnatale giorno 5 neuroni simpatici da topi knockout XIAP erano microiniettati con bovina citocromo c (10 mg / mL) insieme con rodamina-destrano per marcare le cellule iniettate. Le immagini mostrano lo stesso campo di cellule immediatamente dopo l&#39;iniezione (0 ore), o 5 ore dopo la microiniezione del citocromo c (5 ore). Scala grafica, 20 micron.

<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>

Tutte le procedure sperimentali su animali sono state approvate dalla cura degli animali e del Comitato Istituzionale Usa presso l&#39;Università del North Carolina.

La microiniezione del citocromo c direttamente nel citoplasma delle cellule è uno strumento unico e potente che permette di studi di post-regolazione dell&#39;apoptosi mitocondriale. È importante sottolineare che questa tecnica permette l&#39;attivazione diretta di valle apoptosi dei mitocondri, senza l&#39;uso di agenti che causano il danno cellulare o mitocondriale. Anche se questo protocollo si è concentrata sulla microiniezione di citocromo c per gli studi sulla apop...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> DM IRE2 microscopio invertito </td><td> Leica </td><td></td><td></td></tr><tr><td> PC-10 Microiniezione Puller ago </td><td> Narishige </td><td></td><td></td></tr><tr><td> MWO-202 Micromanipolatore </td><td> Narishige </td><td></td><td></td></tr><tr><td> FemtoJet Microinjector </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> A parete sottile Boroscilicate capillare di vetro con microfilamento </td><td> Sistemi AM </td><td> 615000 </td><td> 4 lunghezza cm, 1,00 mm di diametro esterno, 0,75 mm di diametro interno </td></tr><tr><td> Rodamina B isotiocianato-Destrano </td><td> Sigma-Aldrich </td><td> R9379 </td><td> Peso molecolare medio ~ 70.000 Da </td></tr><tr><td> Bovina citocromo c Proteine </td><td> Sigma-Aldrich </td><td> C3131 </td><td></td></tr></tbody></table>

activation of apoptosis by cytoplasmic microinjection of cytochrome c

L&#39;apoptosi, o morte cellulare programmata, è un percorso conservato e altamente regolato con cui le cellule muoiono 1. L&#39;apoptosi può essere attivata quando le cellule incontrano una vasta gamma di sollecitazioni citotossici. Questi insulti avviare cascate di segnalazione che alla fine causano il rilascio del citocromo c dallo spazio intermembrana mitocondriale al citoplasma 2. Il rilascio di citocromo c dai mitocondri è un evento chiave che innesca la rapida attivazione delle caspasi, le proteasi cellulari chiave che alla fine eseguire la morte delle cellule 3-4. Il percorso di apoptosi è regolata a punti a monte ea valle del rilascio del citocromo c dai mitocondri 5. Per studiare la regolazione post-mitocondriale di attivazione delle caspasi, molti ricercatori si sono rivolti a microiniezione diretta citoplasmatica di holocytochrome c (eme-allegato) delle proteine ​​nelle cellule 6-9. Citocromo c è normalmente localizzato nei mitocondri, dove l&#39;attaccamento di un gruppo eme è necessaria per poter attivare l&#39;apoptosi 10-11. Pertanto, per attivare direttamente caspasi, è necessario iniettare la proteina c holocytochrome invece del suo cDNA, perché mentre l&#39;espressione del citocromo c dai costrutti cDNA si tradurrà in targeting mitocondriale e attaccamento eme, sarà sequestrato dalla caspasi citosolico. Così, la microiniezione diretta citosolica purificata di eme-attached proteina citocromo c è un utile strumento per simulare rilascio del citocromo c mitocondriale e l&#39;apoptosi senza l&#39;uso di insulti tossici che provocano danni cellulari e mitocondriali. In questo articolo, si descrive un metodo per la microiniezione di proteine ​​citocromo c nelle cellule, usando il mouse fibroblasti embrionali (MEF) e primario neuroni simpatici come esempi. Anche se questo protocollo si concentra sulla iniezione di citocromo c per le indagini di apoptosi, le tecniche qui riportate può anche essere facilmente adattato per microiniezione di proteine ​​di interesse.

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 questo protocollo, si descrive la microiniezione diretta citoplasmatico del citocromo<em&gt; C</emProteine&gt; in fibroblasti e primario neuroni simpatici. Questa tecnica permette l&#39;introduzione del citocromo<em&gt; C</emProteine&gt; nel citoplasma delle cellule e imita il rilascio del citocromo<em&gt; C</em&gt; Dai mitocondri, che si verifica durante l&#39;apoptosi.

Attivazione di apoptosi da Microiniezione citoplasmatici del Citocromo 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 Views.  University of North Carolina. In questo protocollo, si descrive la microiniezione diretta citoplasmatico del citocromo C in fibroblasti e primario neuroni simpatici. Questa tecnica permette l'introduzione del citocromo C nel citoplasma delle cellule e imita il rilascio del citocromo C Dai mitocondri, che si verifica durante l'apoptosi.

Video: 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.

Intervista: Antigen Presentazione glicolipide da CD1d e il potenziale terapeutico di attivazione delle cellule NKT

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

Ricerca

Biologia

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.

Generazione di Stabile transgenici C. elegans Utilizzando Microiniezione

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

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.

Generazione di transgenici C. elegans Di Trasformazione Biolistic

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.

Analisi della cinetica di esportazione mRNA nucleare in cellule di mammifero da Microiniezione

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.

Analizzando la funzione di GTPasi Small Microiniezione di plasmidi in cellule epiteliali polarizzate

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.

Visualizzazione della funzione respiratoria mitocondriale utilizzando Citocromo C Ossidasi / succinato deidrogenasi (COX / SDH) Doppio etichettatura Istochimica

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.

Sintesi di uno<em&gt; In vivo</em&gt; RM-rilevabile Apoptosis Probe

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.

Singola molecola di imaging del regolamento Gene<em&gt; In vivo</em&gt; Utilizzo di attivazione Cotranslational da Cleavage (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.

Analisi di apoptosi in embrioni di zebrafish da Whole-mount immunofluorescenza per rilevare caspasi 3 attivata

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

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

La stimolazione dei citoplasmatici DNA Sensing Pathways<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...