This work was supported by FIRB-Merit grants RBNE08YFN3_005 and RBNE08HWLZ_012, and the Italian Ministry of Economy and Finance to the CNR for the Project &#8220;FaReBio di Qualit&#224;&#8221;.

1. Bereiten siRNA-Lösungen <ol><li> Resuspendieren 5 nmol von verschlüsselten (Strg) und 5 nmol BRCA2 siRNA getrockneten Pellets in 100 ul RNase-freies Wasser (final siRNA-Konzentration: 50 um). Dies ist die siRNA Lager und muss bei -20 ° C in 10 &amp; mgr; l Aliquots gelagert werden. </li><li> Einen aliquoten von 10 &amp; mgr; l von der siRNA Lager und fügen Sie 40 ul RNase-freies Wasser, das 10 uM siRNA Arbeitslösung zu erhalten. Diese Verdünnung ist bei -20 ° C bis zur Verwendung gelagert werden. HINWEIS: Die siRNA Arbeitslösung sollte nicht unterzogen Einfrieren / Auftauen mehr als 3 mal. </li></ol> 2. Seeding der Menschen Epithelzelllinien <ol><li> Entfernen des Wachstumsmediums aus menschlichen epithelialen Zellinien wachsen als Zellmonoschichten in Gewebekulturplatten in einem befeuchteten Inkubator bei 37 ° C, 5% CO 2. </li><li> Die Zellen in der Platte einmal mit RT PBS (10 ml PBS für eine 100 mm-Platte) zu waschen. </li><li> Um die Zellen von der Platte zu trennen,2 ml 0,25% Trypsin / 0,53 mM EDTA-Lösung und Inkubation 3-5 min, je nach Zelltyp. </li><li> Vor dem Sammeln der abgelösten Zellen neutralisieren Trypsin durch Zugabe von 2 ml komplettem Wachstumsmedium enthaltend 10% fötales Rinderserum enthielt. </li><li> Übertragen Sie die Zellen in ein 15-ml-Tube. </li><li> Zentrifugieren der Zellen bei 320-330 g für 3 Minuten bei 4ºC in einem Schwingbecher-Rotor. </li><li> Resuspendieren des Zellpellets in 5 ml von Antibiotika-freiem Medium. </li><li> Zähle die Zellen unter dem Mikroskop in einer Burker Kammer oder durch Verwendung eines automatischen Zählsystem nach den Anweisungen des Herstellers. </li><li> Seed die Zellen in 6-Well-Platten auf etwa 80% konfluent nach 24 Stunden (0,5-1 x10 6 Zellen in 2 ml Medium für jede Vertiefung) sein. Optimale Zellennummer ausgesät werden können, abhängig von den spezifischen Zellen und der Wachstumsrate variieren. HINWEIS: Antibiotika im Medium Fügen Sie nicht an dieser Stelle, weil Antibiotika negativ mit der Effizienz der transfe störenction. Es ist sehr wichtig, dass die Zellen bei niedrigen Kulturpassagen (&lt;10), um einen hohen Wirkungsgrad der BRCA2 siRNA-Transfektion zu erzielen. </li></ol> 3. Transfektion von Zellen mit siRNA <ol><li> Vierundzwanzig Stunden nach der Plattierung der Zellen, fahren Sie mit der ersten Runde der Transfektion. <ol><li> Verdünnen 6 ul Lipidbasis 10 siRNA spezifischen Transfektionsreagenz in 130 &amp; mgr; l reduziert Serummedium. </li><li> Verdünnen 3 ul von 10 uM siRNA (30 pmol) in 130 &amp; mgr; l reduziert Serummedium. </li><li> Kombinieren Sie die verdünnten siRNA mit der verdünnten Transfektionsreagenz und Inkubation für 5-10 min bei RT. </li><li> Während der Inkubation, entfernen Sie das Medium aus den Zellen und 2 ml frischem Medium bei 37 ° C vorgewärmt (ohne Antibiotika). </li><li> Fügen Sie 250 ul der siRNA-Transfektionsreagenz Komplexe zu den Zellen (dieser Betrag ist für einen einzelnen Vertiefung einer 6-Well-Platte). </li><li> Inkubieren der Zellen in einem befeuchteten Inkubator bei 3776; C mit 5% CO 2. </li></ol></li><li> Nach 24 Stunden, weiter mit der zweiten Runde der Transfektion durch Wiederholung der Schritte 3.1.1-3.1.5 mit folgender Änderung für Schritt 3.1.2: verdünnen 5 ul von 10 uM siRNA (50 pmol) in 130 &amp; mgr; l reduziert Serummedium. <ol><li> Die Zellen für 1-2 Tage bei 37 ° C vor der Analyse. HINWEIS: Zwei Runden Transfektionen und Verwendung eines Hoch siRNA / Transfektionsreagenz Verhältnis in der zweiten Runde sind unerlässlich, um hohe Effizienz der BRCA2-Silencing zu bekommen. </li></ol></li></ol> 4. Lyse Zellen für Immunoblot-Analyse <ol><li> Ansetzen Lysepuffer mit PBS (pH 7,4), enthaltend 1% des nicht-ionischen, nicht-denaturierenden Detergens Octylphenoxypolyethoxyethanol, 5 mM Natriumpyrophosphat, 1 mM Natriumorthovanadat, 10 mM Natriumfluorid, 2 mM Phenylmethylsulfonylfluorid, 10 ug / ml Leupeptin, 10 ug / ml Aprotinin. Halten Sie es auf Eis. </li><li> Entfernen des Mediums von den Zellen und wAsche sie einmal mit kaltem PBS (2 ml / Vertiefung) in der Platte. Um jede PBS Verschleppung zu vermeiden, vollständig zu entfernen von der Platte. </li><li> Zugeben von 200 &amp; mgr; l Lysepuffer zu jedem Well. HINWEIS: Die Zellen müssen auch für andere Tests verwendet werden, trypsinize die Zellen wie in Schritt 2.1-2.6 beschrieben und Resuspendieren der Zellen in einem geeigneten Puffer / Medium gemäß der Anwendung eingesetzt werden. </li><li> Drehen Sie vorsichtig die Platte gleichmäßig zu verteilen das Lyse-Puffer und Inkubation für 15 min bei 4 ° C auf einem Rotator. </li><li> Sammeln des Zelllysats in einem Mikrozentrifugenröhrchen auf Eis mit einem Zellschaber. </li><li> Zentrifuge bei 17.900 xg für 25 min bei 4 ° C und Sammeln des Überstands in ein neues Röhrchen. </li><li> Messung der Proteinkonzentration unter Verwendung eines im Handel erhältlichen Protein-Assay gemß den Herstelleranweisungen. </li></ol> 5. BRCA2 Immunoblotting-Analyse <ol><li> Bereiten Sie den Laufpuffer durch Verdünnung von 50 ml 20x Tris-Acetat SDS Lauf buffer (50 mM Tricin, 50 mM Tris-Base, 0,1% SDS, pH-Wert 8,24) mit 950 ml destilliertem Wasser. </li><li> Vorbereitung der Probe wie folgt: in 15 ug Gesamt-Zelllysat wurden 5 ul 4x Probenpuffer, Lithiumdodecylsulfat bei pH 8,4, 2 ul 10x Probe Reduktionsmittels (0,5 M DTT) und Lyse-Puffer auf ein Endvolumen von 20 ul. Hinweis: Dieses Protokoll ermöglicht den Nachweis von BRCA2 in normalen Zelllinien auch bei Verwendung einer geringeren Menge an Gesamtproteinen (bis auf 5 ug). </li><li> Denaturieren Proben bei 55 ° C für 10 min. Hinweis: Es ist wichtig, auf 55 ° C zu verwenden. Seit BRCA2-Protein ist thermo 11 führen höhere Denaturierung Temperaturen in Einklang Verlust der intakten voller Länge (390 kDa) BRCA2-Protein. </li><li> Die elektrophoretische Kammer gemäß den Anweisungen des Herstellers eingestellt. </li><li> Laden Sie die Proben und 10 ul mit hohem Molekulargewicht vorgefärbten Proteinstandard auf vorgefertigte Gel (Tris-Acetat 8.3%) und bei 120 V für ab laufenout 2 h 12. Stoppen Sie den Elektrophoreselauf bevor die 55 kDa blaue Markierung lässt die vorgefertigte Gel. </li><li> Bereiten Sie den Transferpuffer mit 50 ml 20x Transferpuffer, 100 ml 100% Methanol und 850 ml Wasser. </li><li> Aktivieren PVDF-Membran (0,45 um Porengrße) durch Einweichen in 100% Methanol für 5 min mit destilliertem Wasser gespült und in Transferpuffer äquilibriert für mindestens 5 min. Weichen Sie die Schwämme der Transfervorrichtung in Transferpuffer bei 4 ° C bis zur Verwendung. </li><li> Wenn die Elektrophoreselauf vorbei ist, montieren Sie die Sandwich in der Transferstapel in der folgenden Reihenfolge (von unten nach oben): Drei Schwämme in Transferpuffer gesättigt, ein 3MM Grade Blatt Papier in Transferpuffer, Gels, aktiviert PVDF-Membran, einer gesättigten 3MM Grade Blatt Papier in Transferpuffer gesättigt, drei Schwämme in Transferpuffer 13,14 gesättigt. Platzieren des Stapels in die Vorrichtung und das Transferproteine ​​bei 350 mA für 4 Stunden bei 4 ° C oder bei 180 mA über Nacht bei 4 ° C. HINWEIS: Being BRCA2 ein sehr großes Protein ist, ist es von entscheidender Bedeutung, um die Übertragungszeit von 1 h bis 4 h erstrecken oder über Nacht (wie durch den Hersteller und die meisten Übertragungsprotokolle vorgeschlagen), um eine vollständige Übertragung der hochmolekularen Proteine ​​zu gewährleisten. Darüber hinaus aufgrund der langen Transferzeit ist es wichtig, die Blotting in einem Kühlraum bei 4 ° C durchzuführen, um eine Überhitzung zu minimieren und zu erleichtern Sandwich-Demontage. Halbtrockentransfersysteme sind nicht gut für die Übertragung von großen Proteinen. </li><li> Nach dem Transfer, waschen der Membran mit TBS, blockieren für 1 h bei RT in TBS-Tween 0,25% (TBS-T), das 5% fettfreie Trockenmilch und der Sonde über Nacht bei 4 ° C mit 30 &amp; mgr; l anti-BRCA2 polyklonaler Kaninchen-Antikörper ( 200 ug / ml) in 9 ml TBS-T + 5% fettfreier Trockenmilch verdünnt [1: 300 (v / v) Verdünnung]. </li><li> Dreimal (jeweils 10 Minuten) mit TBS-T Waschen der Membran dann Inkubieren der Filter für 1 h mit 1 &amp; mgr; l Meerrettichperoxidase-konjugiertem anti-Kaninchen Sekundär-Antikörper in 10 ml TBS-T + 5% fettfreier Trockenmilch. Danach waschen Sie die Membran drei Mal (jeweils 10 Minuten) mit TBS-T. </li><li> Führen Immundetektion von ECL zu BRCA2-Proteins aufdecken Verwendung verstärkter Chemilumineszenz (ECL) Western Blotting Nachweisreagenz nach den Anweisungen des Herstellers. Visualisieren immunochemiluminescent Bands auf hochauflösende ECL-Filme. </li><li> Zuführen Immunodetektion mit einem Antikörper für ein Housekeeping-Gen, wie β-Tubulin (55 kDa), auf dem gleichen Filter als Ladekontrolle durch die Verwendung eines monoklonalen Antikörper gegen β-Tubulin einer Verdünnung von 1: 1000 (10 ul von Anti-Tubulin-Antikörper in 10 ml TBS-T + 5% fettfreie Trockenmilch) für 1 h bei RT. Waschen und Inkubieren sekundären Antikörper für BRCA2 (5,10-5,11) beschrieben, mit Ausnahme der Verwendung von Meerrettichperoxidase-konjugiertem Anti-Maus anstelle von Anti-Kaninchen-Sekundärantikörper. </li><li> Erwerben Sie ein digitales Bild der mit immunochemiluminescent Bands beeindruckt Filme und die Bandenintensität von engagierten Bildanalyse-Software zu analysieren (zB., ImageJ). </li></ol>

<ol>
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The authors have nothing to disclose.

Da Keimbahnmutationen des BRCA2-Gen zu einem erhöhten Risiko für verschiedene Krebsarten, einschließlich der weiblichen und männlichen Brustkrebs, Eierstockkrebs, Prostatakrebs, Bauchspeicheldrüsenkrebs, Melanom und 3,4, eine Reihe von Studien durchgeführt, um die biologische Funktion zu verstehen, des BRCA2-Protein. Die meisten dieser Studien sind genetisch-basierte vor allem auf technische Schwierigkeiten bei der Analyse eines Riesenprotein wie BRCA2. Die zum Dämpfen und Analysieren BRCA2-Pr...

Der BRCA2 (Brustkrebsanfälligkeit Gen-2) Gen kodiert für ein Protein, das Tumorsuppressor eine entscheidende Rolle bei der Reparatur von DNA-Doppelstrangbrüchen durch die Regelung der Funktion des Rekombinaseenzym Rad51 1 spielt. BRCA2 ist auch in der Modulation der Transkription und an der Zellzyklussteuerung 2 in Verbindung gebracht. Keimbahnmutationen im BRCA2-Gen induzieren eine autosomal dominant Anfälligkeit für Brust- und Eierstockkrebs bei Frauen und Prostatakrebs bei Männern, als auch Veranlagung zu anderen Krebsarten 3,4. Trotz des erhöhten Risikos bei der Entwicklung von Krebs, BRCA2-Mutationen, die zu unterdrücken oder zu reduzieren BRCA2-Funktion können Krebszellen anfälliger für Chemotherapeutika, die DNA-Schäden verursachen 5-7 rendern. Sporadische Krebserkrankungen weisen eine niedrige Rate von BRCA2-Mutationen (&lt;3%), obwohl reduzierte Mengen an BRCA2-Protein wurden bei einigen Krebsarten festgestellt, was darauf hindeutet, dass die BRCA2-Proteinkann während der Tumorgenese in sporadische Krebserkrankungen durch nicht-Mutationsabhängige Mechanismen 7,8 verloren. Somit ist es wichtig, BRCA2 Funktionen im Zusammenhang mit der Krebsbiologie sowie in anderen biologischen Systemen vollständig verstehen. Ein leistungsfähiges Werkzeug, um neue Funktionen eines Gens in Säugetierzellen zu identifizieren, ist seine Expression unterdrücken. Als ein Beispiel hat Silencing BRCA2-Expression in einer Vielzahl von normalen Epithelzellen kürzlich zur Identifizierung eines neuartigen Funktion BRCA2 als Regulator der anoikis Beständigkeit ein wichtiger Schritt bei der Erfassung von Krebszell invasive und metastatische Fähigkeit 9 geführt. Gen-Silencing kann durch die Einführung in den Zellen entweder small interfering (si) RNA oder short hairpin (sh) RNA-Moleküle gezielt den spezifischen Gens erreicht werden. Die hohe Wirksamkeit der siRNA und seine Benutzerfreundlichkeit machen es die bevorzugte Werkzeug für Silencing-Experimente bei der Gewinnung neuer Erkenntnisse über kritische biologische Prozesse richtet einnd neue therapeutische Targets zu identifizieren. Jedoch kann effizient Zuschlags der Genexpression nicht für alle Gene leicht erreichbar sein und kann sehr unterschiedlich sein, je nach dem Zelltyp. Weil eine Abnahme der intrazellulären Proteinspiegel der relevanteste Phänotyp untersuchten, ist es wichtig, das Gen-Silencing durch Immunoblotting-Analyse des Genprodukts zu quantifizieren. In dieser Hinsicht stellt der BRCA2-Protein eine weitere Herausforderung: ein großes Protein (etwa 390 kDa), haben technische Schwierigkeiten bei herkömmlichen biochemischen Analyse, einschließlich Immunoblotting existieren. Wir berichten hier über ein Protokoll für die effiziente Silencing von BRCA2 in humanen epithelialen Zelllinien und für die schnelle und erfolgreiche Detektion von BRCA2-Protein durch Immunoblotting-Analyse Knockdown optimiert.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>BRCA2 siRNA</td>
			<td>Dharmacon</td>
			<td>L-003462-00</td>
			<td>SMARTpool: ON-TARGETplus BRCA2 siRNA</td>
		</tr>
		<tr>
			<td>Ctrl siRNA</td>
			<td>Dharmacon</td>
			<td>&#160;D-001810-10-05</td>
			<td>ON-TARGETplus Non-targeting Pool</td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX Reagent&#160;</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/13778075">13778075</a></td>
			<td>Transfection Reagent</td>
		</tr>
		<tr>
			<td>OPTI-MEM I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td>Medium for transfection procedure</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate 3-8% 1,5 mm Gel precast</td>
			<td>Life Technologies</td>
			<td>EA0378</td>
			<td>Gel for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample buffer&#160;</td>
			<td>Life Technologies</td>
			<td>NP0007</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Sample Reducing agent (10x)</td>
			<td>Life Technologies</td>
			<td>NP0004</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>NuPAGE Antioxidant</td>
			<td>Life Technologies</td>
			<td>NP0005</td>
			<td>Reagent for protein electrophoresis</td>
		</tr>
		<tr>
			<td>HiMark Pre-stained protein standard</td>
			<td>Life Technologies</td>
			<td>LC5699</td>
			<td>Prestained marker for gel electrophoresis</td>
		</tr>
		<tr>
			<td>XCell SureLoc Mini-Cell&#160; System</td>
			<td>Life Technologies</td>
			<td>EI0002</td>
			<td>Equipment for protein electrophoresis/transfer</td>
		</tr>
		<tr>
			<td>NuPAGE Tris-Acetate SDS Running Buffer</td>
			<td>Life Technologies</td>
			<td>LA0041</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>NuPAGE Transfer Buffer</td>
			<td>Life Technologies</td>
			<td>NP0006-1</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>BRCA2 H-300 rabbit polyclonal antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8326</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>Beta-tubulin monoclonal antibody</td>
			<td>Sigma Aldrich</td>
			<td>T4026-100UL</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Skim Milk powder</td>
			<td>Sigma Aldrich</td>
			<td>70166</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P1379</td>
			<td>Reagent for Western blotting</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico/Femto Chemiluminescent Substrate</td>
			<td>Pierce Thermo Scientific</td>
			<td>34080/34096</td>
			<td>Chemiluminescence system</td>
		</tr>
		<tr>
			<td>PNT1A cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>95012614</td>
			<td>PNT1A human, normal prostate epithelium immortalized with SV40</td>
		</tr>
		<tr>
			<td>Nthy cells</td>
			<td>SIGMA Aldrich (for ECACC)</td>
			<td>90011609</td>
			<td>Nthy-ori 3-1 Cell Line human, thyroid follicular epithelial cells</td>
		</tr>
		<tr>
			<td>Cell Proliferation Kit I (MTT)</td>
			<td>Roche</td>
			<td>11465007001</td>
			<td>Kit for cell proliferation assays</td>
		</tr>
	</tbody>
</table>

silencing of brca2 to identify novel brca2-regulated biological functions in cultured human cells

Inaktivierung des Tumorsuppressor-Proteins BRCA2 und seine Erkennung durch herkömmliche biochemische Analysen stellen eine große technische Herausforderung auf Grund der Größe des menschlichen BRCA2-Protein (etwa 390 kDa). Wir berichten Modifikationen von Standard siRNA-Transfektion und Immunoblotting Protokolle menschlichen BRCA2 endogenen BRCA2-Protein, bzw. in humanen epithelialen Zellinien auszuschalten und zu detektieren. Die wichtigsten Schritte sind, um eine hohe siRNA Transfektionsreagenz Verhältnis und zwei weiteren Runden von siRNA-Transfektion in demselben Experiment. Mit diesen und anderen Modifikationen an dem Standardprotokoll wir mehr als 70% Inaktivierung des menschlichen BRCA2 konsistent zu erreichen, wie durch Immunoblotting mit anti-BRCA2-Antikörpern beurteilt. Zusätzlich Denaturierung der Zelllysate auf 55 ° C anstatt der üblichen 70-100 ° C und anderen technischen Optimierungen des Immunoblotting Verfahren den Nachweis von intakten BRCA2-Protein selbst when sehr geringe Mengen an Ausgangsmaterial zur Verfügung stehen oder wenn BRCA2 Proteinexpressionsniveaus sehr niedrig sind. Effiziente Silencing von BRCA2 in menschlichen Zellen bietet eine wertvolle Strategie zur BRCA2-Funktion in Zellen mit intakten BRCA2, einschließlich Tumorzellen unterbrechen, um neue molekulare Signalwege und zellulären Funktionen, die durch pathogene BRCA2-Mutationen in Tumoren betroffen sein können, zu prüfen. Anpassung dieses Protokoll für die effiziente Schalldämpfung und Analyse von anderen &quot;großen&quot; Proteine ​​wie BRCA2 sollte leicht erreichbar sein.

Bevor mit einer biologischen / biochemischen Test fortfahren, um die Wirkung der BRCA2-Unterdrückung auf Zellfunktionen zu untersuchen, ist der erste Schritt, um die Spezifität des Silencing unter Verwendung eines verschlüsselten siRNA (Non-Targeting-siRNA) Seite an Seite mit BRCA2 siRNA (1A bestätigen ). Es ist wichtig, eine zweite Runde von siRNA-Transfektion mit einem hohen siRNA / Transfektion Verhältnis durchzuführen, um einen hohen Wirkungsgrad der BRCA2-Silencing zu erhalt...

Watch this Scientific Journal Video about Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells at JoVE.com

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

Silencing<em&gt; BRCA2</em&gt; Zum Novel BRCA2-regulierten Biologische Funktionen in kultivierten menschlichen Zellen zu identifizieren

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

silencing-brca2-to-identify-novel-brca2-regulated-biological

Research

JoVE Journal

Biology

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

9.0K Aufrufe.  National Research Council. Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented. 

Serie: Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Live-Imaging von Dense-Core-Vesikel in Primary hippokampalen Neuronen

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Forschung

Biologie

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolierung von Ribosom gebunden Nascent Polypeptide<em&gt; In-vitro-</em&gt; Identifizieren Translational Pause Standorten entlang mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Eine quantitative Assay zur Protein Studie: DNA-Wechselwirkungen, Discover Transkriptionsregulatoren der Genexpression, und identifizieren Novel Anti-Tumor-Wirkstoffe

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualisierung des endoplasmatischen Reticulum Subdomains in kultivierten Zellen

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Capture Compound Mass Spectrometry - Ein leistungsfähiges Tool zum Identifizieren Novel c-di-GMP Effektorproteinen

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Effiziente Erzeugung Menschen induzierten pluripotenten Stammzellen aus menschlichen Körperzellen mit Sendai-Virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA Screening zu Ubiquitin und Ubiquitin-ähnlichen System Regler der biologischen Stoffwechselwege in kultivierten Säugerzellen identifizieren

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nephrotoxin Mikroinjektion in Zebrabärbling akuten Nierenschädigung zu Modell

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Hochauflösende Zeitraffer-Imaging und automatische Analyse von Dynamik der Mikrotubuli in lebenden menschlichen Endothelzellen aus der Nabelvene

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Eine grafische Benutzeroberfläche für die softwaregestützte Verfolgung der Proteinkonzentration in dynamischen zellulären Vorsprüngen

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Silencing

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