We thank Yumeng (Helen) Xia (Rutgers University) for manuscript editing. The work on BASL polarity formation is supported by grants from the U.S. National Institute of General Medical Sciences to J.D. (R01GM109080) and Rutgers University.

1. Costruzione plasmide <ol><li> Generare i costrutti di clonazione tecnologia come descritto in precedenza 1,6. <ol><li> In primo luogo utilizzare un DNA polimerasi ad alta fedeltà con primer adeguati per amplificare le sequenze codificanti di BASL, YDA e MPK6 e sub-clonare in vettori di ingresso. Trova il protocollo dettagliato in 7. </li><li> Per generare le versioni negative dominanti (rispettivamente 8 e DNmpk6 DNyda 4,) MPK6 e YDA, utilizzare i plasmidi di entrata di MPK6 e YDA come modello e introdurre le mutazioni puntiformi con un metodo mutagenesi sito-9. </li></ol></li><li> Per costruire i costrutti per saggi transienti tabacco, condurre una serie LR (Attl x attr ricombinazione) reazioni (procedura dettagliata 7) per integrare BASL in pH35CG 10, DNyda e DNmpk6 in pXNGW e pXCGW vettori 11, rispettivamente. <ol><li> Confermare i plasmidi risultanti di digestione enzimatica e sequenziamento. Una volta che sUccessful, trasformare i costrutti binari nel Agrobacterium tumefaciens ceppo GV3101 12 e farle crescere su piastre di selezione a 28 ° C per 2 - 3 giorni. </li></ol></li></ol> 2. Preparazione della soluzione Agrobacterium Infiltration <ol><li> Seminare alcune colonie di Agrobacterium appena trasformati in 10 ml di Luria-Bertani (LB) media con antibiotici appropriati (Gentamicina 25 mg / ml, Rifamicina 25 mcg / ml, Spectinomicina 100 ug / ml per pH35CG, pXNGW e pXCGW costruisce, le stesse concentrazioni di gentamicina e kanamicina Rifamicina con 50 mg / ml per P19 (che esprimono le proteine ​​contro silenziamento transgenico) 13. </li><li> Grow culture Agrobacterium in C shaker 28 ° alla velocità di 200 rpm per circa 16 ore e quindi misurare la OD 600 (stimato crescere dell&#39;1,5 - 1.8) (Figura 2A-B). Centrifugare le culture a 2.500 xg per 10 min. Risospendere e wash i pellet cellulari con 10 mM di MgCl 2 (la soluzione di infiltrazione). </li><li> Spin giù le culture di nuovo e ri-sospendere i pellet con la quantità appropriata della soluzione di infiltrazione per raggiungere la finale OD 600 = 0,5 (Figura 2C-D). Prima di infiltrazione piante, lasciare le miscele di cella a temperatura ambiente per 1-3 ore. Questo passaggio consente di mantenere l&#39;omeostasi cellulare. </li></ol> 3. Impianto di tabacco e Foglia Iniezione <ol><li> Utilizzare le piante di tabacco (Nicotiana benthamiana), cresciuti 4-5 settimane di età in una camera di crescita delle piante standard (23 ° C con cicli di 16 ore-luce / 8 ore buio) 14 per l&#39;iniezione foglia. Nota: In questa fase, le piante di tabacco in genere hanno 6 foglie (2 grandi, 2 medie e 2 piccole). Le due foglie medie sono il più ottimale per l&#39;iniezione del foglio (i due grandi sono troppo vecchi, mentre i due piccoli sono spesso troppo giovane). </li><li> Prima del giorno di infiltrazione, innaffiare il Tobacpiante co per saturare il terreno. </li><li> Il giorno infiltrazione, pre-incubare le piante al buio per 1 - 3 ore. Questa fase consente stomi nell&#39;epidermide per aprire ampiamente, che facilita notevolmente l&#39;Agrobacterium quando penetra nelle cellule epidermiche foglia. </li><li> Usando nastri di colore etichettate con date, bandiera le foglie di essere infiltrati. Selezionare le aree da infiltrati con un pennarello nero spesso (facoltativo; le zone infiltrate sono visibili dopo l&#39;infezione.). </li><li> Prendere volumi uguali di Agrobacterium risospensione e mescolarli in un 100 x 15 mm Petri delicatamente vorticoso (Figura 2E). Nota: Nel nostro caso, abbiamo mescolato 1 ml di p19 con 1 ml di CFP-BASL, 1 ml di DNyda-nYFP e 1 ml di DNmpk-cYFP. </li><li> Nel processo di infiltrazione, riempire una siringa da 3 ml di ago con la miscela infiltrazione (1 - 2 ml) e spingere sul lato inferiore (abaxial) della foglia di tabacco (Figura 2F). Mentre infiltrazione, è utilead usare una mano per spingere e l&#39;altra mano per sostenere delicatamente il lato superiore (adaxial, contro la forza di spinta). Nota: Una volta che la miscela di infiltrazione viene iniettato con successo, le zone infette saranno facilmente riconoscibili nell&#39;epidermide. Tuttavia, l&#39;infiltrazione potrebbe non riuscire a causa di iniezione dura (danni ai tessuti) o spinta insufficiente (nessuna penetrazione liquido). Per ogni test, si consiglia almeno due iniezioni in due foglie indipendenti (da piante diverse). </li><li> Posizionare le piante infette di nuovo nella camera di crescita. In generale, le espressioni di proteine ​​sono rilevabili dopo 48 ore dopo l&#39;infiltrazione. </li></ol> 4. confocale Imaging <ol><li> A 48 ore dopo l&#39;infiltrazione, usare una perforatrice per asportare un disco foglia dalla zona di iniezione (normalmente 3 - 4 dischi / zona possono essere prodotte) (Figura 2G). Utilizzare le pinzette per trasferire delicatamente i dischi di foglie di una diapositiva (il lato abaxial verso l&#39;alto) e montare loro con gocce d&#39;acqua. Evitare di premere troppo harD sul vetrino perché spremuti / cellule danneggiate non mostrerà ben al microscopio confocale (Figura 2H). </li><li> Prima confocale, la scansione dei campioni montati su un microscopio composto epi-fluorescenza per controllare i livelli di espressione. Nota: In base alla nostra esperienza, le cellule che mostrano livelli di espressione intermedi sono meglio rappresentati al microscopio confocale. </li><li> Inizia con la (1.3 NA) obiettivo 40X su un microscopio confocale. Regolare la slitta nella posizione desiderata in modo che le cellule che esprimono livelli medi di proteina fluorescente rimangono al centro. Gli spettri di eccitazione / emissione per PCP e YFP sono 458 nm / 480-500 nm e 514 nm / 520-540 nm, rispettivamente. </li><li> Attivare il pulsante &quot;Live&quot; per visualizzare in anteprima le immagini quindi regolare l&#39;intensità del laser e intelligente guadagno per il rapporto di fluorescenza intensità / rumore più equilibrata. Per migliorare la qualità delle immagini, aumentare pixel scansione e / o medio cornice / linea. Infine, regolare la manopola di messa a fuoco a reach il piano focale mediana e fare clic su &quot;Capture&quot; per acquisire l&#39;immagine. Nota: La PCP e le immagini YFP possono essere catturate in sequenza o simultaneamente controllando o disattivare l&#39;opzione di modalità di scansione &quot;sequenziale&quot;, rispettivamente. </li><li> Quando Z-proiezione è desiderato per la visualizzazione in profondità delle celle, selezionare la modalità di imaging &quot;xyz&quot;, definire la profondità della portata di scansione (10 - 15 micron) e raccogliere le immagini dalla cima al fondo della cellula bersaglio . Compilare le immagini ottenute con il software Leica facendo clic destro sul file di immagine per selezionare &quot;rinomina&quot; e esportare le immagini come file TIFF. </li><li> Immagine 30 - 40 cellule per l&#39;analisi di quantificazione. </li></ol> Processing 5. Immagine e analisi Quantificazione <ol><li> Utilizzare il software Fiji (http://fiji.sc/Fiji) per elaborare le immagini e condurre la quantificazione. <ol><li> Tracciare i grafici di intensità di fluorescenza. <ol><li> Per visualizzare meglio se l&#39;espressione CFP / YFP èuniformemente distribuito, usare Fiji per dimostrare la forza della PCP e YFP segnale lungo la periferia delle cellule. Per raggiungere questo obiettivo, primo lancio Fiji quindi selezionare &quot;File&quot; e &quot;Open&quot; per avviare l&#39;immagine. Fai clic su &quot;linea&quot; nel menu degli strumenti e selezionare &quot;linea segmentata&quot; facendo clic destro. Decidere la regione di interesse e tracciare una linea lungo la periferia cellulare per rintracciare il PCP o il segnale YFP (figura 3A-C). Nota: Fiji misura il valore assoluto intensità di fluorescenza lungo le linee segmentate. </li><li> Selezionare il menu a discesa &quot;Analizza&quot; e &quot;profilo di stampa&quot; per generare un grafico con valori di posizione e l&#39;intensità che rappresentano i livelli di CFP / YFP lungo la linea segmentata. Se lo si desidera, duplicare i valori di intensità (selezionando il menu &quot;Copia&quot;) in Excel o altri software di grafica per generare grafici di intensità paragonabile. </li></ol></li><li> Quantificare grado di polarità. <ol><li> Raccogliere circa 20 rapprecellule tativi da 3 esperimenti replicati per valutare il grado di polarità della PCP e YFP in cellule di tabacco. Calcolare il grado di polarità sulla base del rapporto tra l&#39;intensità di fluorescenza (definita come H) sull&#39;intensità di fluorescenza bassa (definita come L). Nota: Il metodo di quantificazione è spiegato nella figura 3. <ol><li> Per misurare i valori di H e L, tracciare una linea segmentata lungo la regione interessata alla periferia delle cellule (sopra descritto), fare clic su &quot;Analizza&quot; dal menu a discesa, quindi selezionare &quot;Measure&quot;. Quando una finestra &quot;Risultati&quot; si apre, usare &quot;Mean&quot; valori per eseguire quantificazione. </li><li> Dalle cellule che mostrano espressione relativamente uniforme della PCP e YFP, ad esempio, CFP-BASL (figura 3A) o YFP completati dal co-espressione di DNyda-nYFP e DNmpk6-cYFP (Figura 3B), ottenere i valori di H e L, misurando due segmenti selezionati in modo casuale periferiche con uguale lunghezza. </li> &lt;li&gt; Dalle cellule espositrici evidente accumulo polare di proteine ​​fluorescenti, in questo caso, la co-espressione di CFP-BASL, DNyda-nYFP e DNmpk6-cYFP (Figura 3C), raccoglie i valori di H delle regioni periferiche con i segnali di fluorescenza arricchiti e le LS da regioni con nessun accumulo bassa / fluorescenza. </li><li> Pool i rapporti risultanti di H / L da 20 celle insieme e di prova per la distribuzione normale. 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Uso generale e possibili modifiche del sistema di espressione transitoria Tabacco per trasduzione del segnale: sovraespressione di proteina fluorescente (FP) -tagged proteine ​​dell&#39;epidermide tabacco è stato utilizzato con successo per l&#39;esame veloce di proteine ​​localizzazione subcellulare in cellule vegetali. Tuttavia, studiando la distribuzione di segnalazione dinamica del complesso proteico in Planta è un argomento difficile a causa di contesti subcellulari complicate, i...

Le proteine ​​interagiscono all&#39;interno di un intricato gerarchici complessi di rete e di forma che giocano un ruolo fondamentale in quasi tutti i processi biologici che avvengono in un ambiente cellulare imprevedibile. Tuttavia, dallo sviluppo pianta risposte di crescita, c&#39;è stata una mancanza di strumenti utili che possono non solo rapidamente, ma anche identificare e sorvegliare efficacemente questi eventi di segnalazione dinamici a livello subcellulare. Il sistema di espressione della proteina transitoria tabacco epidermide foglia è attraente vantaggi per la visualizzazione di proteine ​​fluorescenti nelle cellule viventi. Questo sistema fornisce semi in vivo condizioni che permettono la modifica della proteina post-traslazionale e rapido esame di localizzazione delle proteine. Esaminando il segnale YFP completato, il test biomolecolare complementazione fluorescenza (BiFC) informa la possibilità di interazione proteina-proteina nelle cellule vegetali. Rispetto ad altri metodi, ad esempio, il lievito-due ibridi (Y2H) e co-immunoprecipitation (Co-IP), BiFC fornisce anche un potente mezzo di visualizzazione comparti in cui possono verificarsi interazioni proteina-proteina a livello subcellulare. Poiché la divisione cellulare asimmetrica (ACD) mantiene staminali popolazione cellulare durante la generazione di nuovi tipi di cellule per la formazione del tessuto / organo, è un meccanismo indispensabile per promuovere multicellularità eucarioti. Arabidopsis sviluppo degli stomi è stato utilizzato come sistema modello per lo studio ACD nelle piante. La cellula precursore, cellula madre meristemoid, divide in modo asimmetrico per produrre due differenti cellule figlie, una meristemoid (subisce staminali divisioni cellulari come prima di terminare in un paio di cellule di guardia) e una cella stomatica lignaggio terra (SLGC) (può dividersi e differenziarsi in una cella marciapiede), rispettivamente (Figura 1). In stomatal ACD, la rottura nuova proteina di asimmetria nel lignaggio stomatica (BASL) è polarizzato premitotically guidare asimmetrie divisione, che incLude asimmetria fisica e il destino delle cellule asimmetria 1. Una cascata MAPK composto dal MAPKKK Yoda e le MAPKs, MPK3 e 6 è centrale per la divisione degli stomi patterning e l&#39;adozione destino 2,3, 4,5. Recentemente, Zhang et al. collegato la proteina BASL polarità della via di segnalazione YDA-MAPK in Arabidopsis stomi ACD 6. Il percorso canonico YODA-MAPK, attraverso MPK3 / 6, fosforila BASL e attiva la sua polarizzazione. Funzioni BASL fosforilata da impalcatura e recluta YODA (YDA) e MPK3 / 6 per formare un complesso proteico e concentrano la segnalazione alla corteccia cellulare 6. Polarizzazione dei componenti MAPK e l&#39;anello di retroazione positiva tra BASL e il percorso YDA-MAPK rappresenta un nuovo meccanismo per la polarizzazione delle proteine ​​nelle cellule vegetali. Segnalazione MAPK Localmente arricchita si ipotizza essere strettamente legata alla differenziazione destino della cellula in stomatica ACD 6 (Figura 1). Uno dei key dati sperimentali che ha sostenuto questo modello è venuto dai saggi di tabacco che hanno dimostrato la ridistribuzione spaziale di MAPK indotte mediante l&#39;espressione di BASL 6. In generale, non è facile controllare dove si verifica segnalazione MAPK perché molecole MAPK erano spesso trovano ovunque all&#39;interno di una cellula. In questo studio, abbiamo utilizzato il sistema split-YFP per visualizzare l&#39;interazione tra la chinasi monte e quelli a valle di suggerire dove avviene il relè di segnalazione. Abbiamo esteso ulteriormente l&#39;utilizzo del sistema BiFC dal co-esprimendo una terza proteina (PCP-tagged), con la scissione YFP due (suggestiva di interazione proteina-proteina) di visualizzare se e come il YFP integrata potrebbe essere spazialmente modulata dal co espresso proteina CFP. In questo modo, abbiamo dimostrato che la co-espressione di CFP-BASL indotta riorganizzazione spaziale delle interazioni tra YDA e MPK6, da una distribuzione uniforme di patterning polarizzato in corteccia cella del epidermide tabaccocellule al. Questo sistema ha quindi il potenziale per essere sviluppato per il monitoraggio di eventi di segnalazione dinamici in cellule vegetali, in condizioni in cui le cellule sono sfidati dagli stimoli interni o esterni (ad esempio, co-espressione di altre proteine, applicazione di prodotti chimici, di attacco patogeno o cambiamenti ambientali, ecc. ).

<table>
	<tbody>
		<tr>
			<td>Phusion DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR/D/TOPO</td>
			<td>Life Technologies</td>
			<td>K2400-20</td>
			<td></td>
		</tr>
		<tr>
			<td>QuickChange II XL Site-directed Mutagenesis Kit</td>
			<td>Agilent Technology</td>
			<td>200521</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>AMRESCO</td>
			<td>J106-2KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>AMRESCO</td>
			<td>J673-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Sigma-Aldrich</td>
			<td>G3632-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Sigma-Aldrich</td>
			<td>R3501-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td>K4000-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Sigma-Aldrich</td>
			<td>S4014-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>25x75mm Slide</td>
			<td>VWR</td>
			<td>16004-382</td>
			<td></td>
		</tr>
		<tr>
			<td>24x50 mm Cover glass</td>
			<td>VWR</td>
			<td>48393-241</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td>Leica&#160;</td>
			<td>TCS SP5 II</td>
			<td>&#160;LAS AF Lite software</td>
		</tr>
		<tr>
			<td>40 x objective lens</td>
			<td>Leica</td>
			<td>HCX&#160;Plan APO&#160;</td>
			<td>HCX&#160;Plan APO&#160;, NA 1.30</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>SORVALL 6+</td>
			<td></td>
		</tr>
		<tr>
			<td>Hole puncher</td>
			<td>Staples</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml falcon tubes</td>
			<td>Genesee Scientific</td>
			<td>21-108</td>
			<td></td>
		</tr>
		<tr>
			<td>No. 5 Forceps</td>
			<td>Canemco &#38; Marivac</td>
			<td>205EQA-Spec</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging spatial reorganization of a mapk signaling pathway using the tobacco transient expression system

Visualizzazione di eventi di segnalazione dinamici in cellule vive è stata una sfida. Abbiamo ampliato un sistema di espressione transiente stabilito, il biomolecolare complementazione fluorescenza (BiFC) test in cellule epidermiche tabacco, dall&#39;interazione test proteina-proteina per monitorare la distribuzione spaziale della trasduzione del segnale nelle cellule vegetali. In questo protocollo, abbiamo utilizzato il test BiFC per dimostrare che l&#39;interazione e la segnalazione tra la Arabidopsis MAPKKK Yoda e MAPK6 si verificano a livello della membrana plasmatica. Quando il BASL proteina scaffold è stato co-espresso, l&#39;interazione YODA-MAPK ridistribuita e spazialmente co-polarizzato con CFP-BASL. Questo sistema di espressione del tabacco modificato consente per l&#39;esame rapido di segnalazione di localizzazione e dinamici cambiamenti (meno di 4 giorni) e in grado di ospitare più di colori proteina fluorescente (almeno tre). Abbiamo anche presentato metodi dettagliati per quantificare la distribuzione delle proteine ​​(localizzazione spaziale asimmetrica, o &quot;polarizzazione&quot;;) In cellule di tabacco. Questo avanzato sistema di espressione del tabacco ha un potenziale per essere utilizzato ampiamente per il test rapido di eventi di segnalazione dinamici in cellule vegetali dal vivo.

Per prevenire la morte cellulare indotta dalla segnalazione MAPK iperattivo, le chinasi versioni inattive di YDA (DNyda) e MPK6 (DNmpk6) sono stati utilizzati nel saggio di co-espressione in cellule di tabacco. Né l&#39;interazione tra DNyda e DNmpk6 rivelato da BiFC né CFP-BASL stesso generato modello di distribuzione non uniforme (Figura 3A-B). Tuttavia, quando CFP-BASL stato introdotto nella coppia interazione DNyda-DNmpk6, sia CFP e YFP segnali sono stati ridistrib...

Watch this Scientific Journal Video about Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System at JoVE.com

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Imaging Riorganizzazione spaziale di una via di segnalazione MAPK Utilizzando il sistema transitorio Expression Tabacco

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

The overall goal of this procedure is to visualize dynamic spatial distribution of protein

imaging-spatial-reorganization-mapk-signaling-pathway-using-tobacco

Research

JoVE Journal

Biology

9.7K Views.  Rutgers, The State University of New Jersey. The overall goal of this procedure is to visualize dynamic spatial distribution of protein

Video: Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Ricerca

Biologia

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Sezioni incluse in paraffina e surgelati di Drosophila Adulti Muscoli

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

Visualizzazione di Caenorhabditis Elegans Strutture cuticola Utilizzando il Dye lipofili Vital, Dii

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Un alto contenuto di flusso di lavoro di imaging per studiare GRB2 Centri di segnalazione di espressione clonazione

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Agroinfiltration efficiente di impianti di alto livello espressione transiente di proteine ​​ricombinanti

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

La quantificazione temporale di espressione MAPK indotta in cellule di lievito singole

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Espressione transiente di proteine ​​da parte idrodinamica Gene Consegna in topi

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Misurazione del tasso metabolico in<em&gt; Drosophila</em&gt; Utilizzando respirometria

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.