Die vorliegende Arbeit wurde in Teilen unterstützt durch von den Canadian Institutes of Health Research / Fragile X Research Foundation of Canada Partnerschaftsprogramm zu RD Finanzierung der Jerome Lejeune Stiftung RD, dem Interdisziplinären Zentrum für Klinische Forschung der Universität Erlangen-Nürnberg auf RD und von der Deutschen Forschungsgemeinschaft zu RD und RE. Die Autoren möchten Ingrid Zenger für die technische Unterstützung bei der Zellkultur Wartung sowie Prof. M. Wegner zur Herstellung eines pCMV5-FLAG-Vektor zur Verfügung zu danken. Die Autoren wollen weiter besonders Nadja Schroeder für die hilfreiche Unterstützung bei der Videoaufnahmen danken.

1. Stammlösungen <ol><li> Resuspendieren des lyophilisierten aktiven Motiv Pulver in sterilem H 2 O auf eine Endkonzentration von 2 ug / ul. Tippen Sie vorsichtig zum Mischen. </li><li> Bereiten Sie kleine Teilmengen ( zum Beispiel 12 &amp; mgr; l jeweils 2 ul pro Reaktion benötigt werden) und speichern sie bei -20 ° C. </li></ol> 2. Herstellung von Zellen <ol><li> Seed Säugerzellen wie HEK293-Zellen oder primären Neuronen auf einer Platte mit 24 Vertiefungen in 500 &amp; mgr; l Wachstumsmedium mit Antibiotika. HINWEIS: Wenn Neuronen verwenden, die Zellen bei geringer Dichte Saatgut und verwenden Sie nur die beiden mittleren Reihen der 24-Well-Platte. Jede Vertiefung wird also ein leeres Gegenstück, das Medium während der Inkubation (Schritt 4.2) zu beherbergen. Wenn Neuronen Handhabung, immer darauf achten, dass die Zellen nicht außerhalb des Inkubators gehalten länger als ein paar Minuten. </li><li> Kultur die Zellen bei geeigneten Bedingungen (befeuchteten, 5% CO 2 und 37 ° C) , bis die Zellen app sind. 50%konfluent. HINWEIS: Wenn Neuronen verwenden, Kultur die Zellen, bis die gewünschte Entwicklungsstadium erreicht ist und App ändern. 25-50% des Mediums zweimal pro Woche. Medium: Neurobasal Medium (enthaltend 1x B27, 5 mM L-Glutamin und 1x Penicillin und Streptomycin; Vergleichen mit ref 16). </li></ol> 3. Chariot Komplexbildung <ol><li> Pro Reaktion, 0,1-2 ug des POI oder den entsprechenden Antikörper sowie das Kontrollprotein oder Kontrollantikörper, die jeweils in 50 ul PBS verdünnen. HINWEIS: Die Technik arbeitet auch mit makromolekularen Komplexen , bestehend aus Vor-gebundenen primären und sekundären Antikörpern (cp &#39;Repräsentative Ergebnisse&#39;, Fig . 1). Mix und Spin. </li><li> Für jede Probe verdünnen 2 ul Chariot Stammlösung in 50 ul PBS mit getrennten Röhren, um die Selbstassoziation von Chariot zu vermeiden. Mix und Spin. </li><li> Übertragen Sie die verdünnte POI oder Antikörper (Schritt 3.1) an die Chariot Verdünnung durch pipettierenting. Mix und Spin. </li><li> Inkubieren der Mischung für 30 min bei Raumtemperatur (Chariot-POI-Komplexe bilden). </li></ol> 4. Transfektion von Zellen <ol><li> Verdünnen Sie die Chariot-Komplexe (Schritt 3.4) in 100 &amp; mgr; l vorgewärmten Wachstumsmedium (37 ° C, ohne Zusätze). Entfernen Sie das Medium und waschen Sie einmal die Zellen vorgewärmt 1x PBS. HINWEIS: Wenn Neuronen verwenden, sollten Sie das Medium nicht zu verwerfen, wird es wieder verwendet werden. Halten Sie es bei 37 ° C. Zum Waschen verwenden PBS , das 0,5 mM MgCl 2 und CaCl 2. Vorschlag: halten das Medium in leeren Brunnen, während die Platte Inkubation (Schritt 4.5). </li><li> Tragen Sie die Chariot-Komplex-Lösung (Schritt 4.1) zu den Zellen und die Zellen schaukeln sanft eine gleichmäßige Verteilung der Lösung zu gewährleisten. Wachsen die Zellen unter Standardbedingungen (Schritt 2.2) für 1 Stunde. Hinzufügen Serum bis zu einer Endkonzentration von 10%. Wenn Neuronen verwenden, fügen Sie das Medium aus Schritt 4.1. Wachsen die Zellen für 1 bis 2 weitere Stunden. Beachten Sie dasZeit optimale Inkubationszeit hängt von der Größe und den Eigenschaften der Ladung und müssen empirisch eingestellt werden. Die folgenden Vorschläge können als Richtlinie dienen: Für Peptide, 1 h in der Regel ausreichend ist, die für Proteine ​​1-2 Stunden empfohlen werden, für Antikörper 2 h und für die Transfektion von Neuronen mit Antikörpern 4 Stunden. </li><li> Prozess Zellen für die Analyse wie üblich. Bitte beachten Sie: die Technik, mit Experimenten ist kompatibel mit Fixierung Protokolle sowie Live-Bildgebung. </li></ol> 5. Imaging <ol><li> Mit Hilfe eines Laser-Scanning-Mikroskop, nehmen Sie z-Stapeln von Zellen und / oder Zellräumen von Interesse etwa 0,25-0,5 &amp; mgr; m Abstand verwenden. Die genaue Schichtabstand hängt von der Größe der Struktur rekonstruiert werden und muss individuell bestimmt werden. </li></ol> 6. Wiederaufbau <ol><li> In den folgenden Schritten, verwenden Sie die Esc Boden mehrere Objekte zwischen Auswahl und Navigation, Cntr zu wechseln auswählen, indem Sie on sie und Shift-Taste zu schneiden Objekte (vgl. Schritt 6.10). </li><li> Öffnen Sie die LSM-Datei in Imaris. </li><li> Mit der Display-Einstellung für jeden Kanal, Kontrast das Bild, bis die hellsten Strukturen Sättigung erreichen. Bitte beachten Sie, dass diese Einstellung die Oberflächenkonstruktion nicht beeinflussen, ist es nur der Experimentator zu unterstützen dient das Bild in Schwellwertbildung. </li><li> Klicken Sie auf das Hinzufügen neuer Oberflächen-Symbol. Ein Assistent wird geöffnet. </li><li> Wählen Sie: Segment nur eine Region of Interest. Fahren Sie mit dem nächsten Schritt. </li><li> Passen Sie die Ränder der Auswahlbox Ihr Objekt von Interesse zu passen. Bitte beachten Sie, dass das Bild 3 Dimensionen hat und dass die Auswahl muss auch in der z-Ebene eingestellt werden. Wenn die rechteckige Form der Auswahlbox nicht das Objekt von Interesse richtig passen sollte, schalten Sie das Objekt und / oder das Feld vergrößern, bis alle erforderlichen Strukturen eingebettet sind. Unerwünschte Objekte können später entfernt werden (vgl. Schritt 6.10). Vorgehen. </li><li> Wählen Sie die entsprechende channel. Die Fläche Detailebene oder Kugeldurchmesser sollten die Strukturen werden rekonstruiert übereinstimmen eingestellt werden. In Abhängigkeit von den Signaleigenschaften, entweder absolute Intensität oder Local Contrast verwendet werden. Im Allgemeinen arbeitet Lokaler Kontrast besser für diffuse Signale. In jedem Fall müssen die Einstellungen für jedes Signal oder interessierende Struktur separat eingestellt werden, respectively. </li><li> Mit dem Thresholding Werkzeug, rekonstruieren die Struktur von Interesse. </li><li> Füllen Sie die Rekonstruktion, indem Sie auf den grünen Pfeil unten klicken. </li><li> Stellen Sie das Objekt weiter von dem Stift-Werkzeug Oberflächen zu schneiden und zu entfernen, wie erforderlich. Es ist nur möglich, in Nord-Süd-Richtung zu schneiden, also kippen das Bild, um das gewünschte Objekt zu schneiden. </li><li> Erhalten Messungen von Volumen, Fläche und Intensitäten für jede Fläche durch die Auswahl Statistiken, detaillierten Statistiken und Durchschnittswerte. </li><li> (Optional Schritt) farbcodierte Statistiken zu beobachten (vgl. Abbildung 2E </strong&gt; und F), wählen Sie die Registerkarte Farbe der entsprechenden Oberfläche und markieren Sie &quot;Statistik Coded&quot; anstelle von &quot;Base&quot;. Mehrere Optionen werden angezeigt. </li></ol>

<ol>
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M., Vendruscolo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+on+the+edge:+a+link+between+gene+expression+levels+and+aggregation+rates+of+human+proteins.">Life on the edge: a link between gene expression levels and aggregation rates of human proteins.</a> Trends Biochem Sci. 32, 204-206 (2007).</li><li>Schwanhausser, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+quantification+of+mammalian+gene+expression+control.">Global quantification of mammalian gene expression control.</a> Nature. 473, 337-342 (2011).</li><li>Uchino, K., Ochiya, T., Takeshita, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+therapeutics+and+applications+of+microRNAs+in+cancer+treatment.">RNAi therapeutics and applications of microRNAs in cancer treatment.</a> Jpn J Clin Oncol. 43, 596-607 (2013).</li><li>Li, T., Wu, M., Zhu, Y. Y., Chen, J., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+RNA+interference-based+therapeutics+and+application+of+multi-target+small+interfering+RNAs.">Development of RNA interference-based therapeutics and application of multi-target small interfering RNAs.</a> Nucleic Acid Ther. 24, 302-312 (2014).</li><li>DeVincenzo, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise,+pitfalls+and+progress+of+RNA-interference-based+antiviral+therapy+for+respiratory+viruses.">The promise, pitfalls and progress of RNA-interference-based antiviral therapy for respiratory viruses.</a> Antivir Ther. 17, 213-225 (2012).</li><li>Fellmann, C., Lowe, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+RNA+interference+rules+for+silencing.">Stable RNA interference rules for silencing.</a> Nat Cell Biol. 16, 10-18 (2014).</li><li>Morris, M. C., Depollier, J., Mery, J., Heitz, F., Divita, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peptide+carrier+for+the+delivery+of+biologically+active+proteins+into+mammalian+cells.">A peptide carrier for the delivery of biologically active proteins into mammalian cells.</a> Nat Biotechnol. 19, 1173-1176 (2001).</li><li>Derlig, K., Gie&#223;l, A., Brandst&#228;tter, J. H., Enz, R., Dahlhaus, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Characterisation+of+Simiate,+a+Novel+Protein+Linked+to+the+Fragile+X+Syndrome.">Identification and Characterisation of Simiate, a Novel Protein Linked to the Fragile X Syndrome.</a> PLoS One. 8, e83007 (2013).</li><li>Derlig, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simiate+is+an+Actin+binding+protein+involved+in+filopodia+dynamics+and+arborisation+of+neurons.">Simiate is an Actin binding protein involved in filopodia dynamics and arborisation of neurons.</a> Frontiers in Cellular Neuroscience. 8, (2014).</li><li>Loudet, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-covalent+delivery+of+proteins+into+mammalian+cells.">Non-covalent delivery of proteins into mammalian cells.</a> Org Biomol Chem. 6, 4516-4522 (2008).</li><li>Nagel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+microspheres+as+protein+transduction+reagents.">Polymeric microspheres as protein transduction reagents.</a> Mol Cell Proteomics. 13, 1543-1551 (2014).</li><li>Selim, A. 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The authors have nothing to disclose.

Hier stellen wir ein Protokoll die Bedeutung von Protein-Expressionsniveaus in Kontrolle zellulärer Funktionen in einer Dosis angetrieben Weise zu studieren. Das beschriebene Protokoll ermöglicht eine Feinabstimmung Manipulation der Proteinexpression in verschiedenen Säugetierzelltypen, einschließlich Hippocampusneuronen, damit detaillierte Studien der Proteinfunktion auf zellulärer Ebene zu erleichtern. Obwohl die RNA - Interferenz eine bekannte Strategie POIs repräsentiert herunterre...

Eine strenge Kontrolle der Proteinexpression ist für jeden lebenden Organismus seine eigene Entwicklung als auch zu befehlen, als auf Umweltsignale zu reagieren. Daher wurde eine Vielzahl von Mechanismen erfunden während der Evolution genau das Expressionsniveau jedes von dem App kodierten Proteins regulieren. 20.000 Gene in jeder eukaryotischen Zelle zu einem bestimmten Zeitpunkt ihres Lebens existiert. Es findet in den verschiedenen Phasen der Proteinproduktion reichen die Regulationsmechanismen aus dem Management der Chromatinstruktur, Transkription und RNA auf die Richtung der posttranslationalen Proteinmodifikationen Handhabung, Transport und Abbau. Es ist daher nicht verwunderlich, dass Störungen in der zugrunde liegenden molekularen Maschinerien und veränderten Proteinexpressionsniveaus haben mit verschiedenen Krankheiten wie Krebs oder geistiger Behinderung in Verbindung gebracht worden. Tatsächlich an der herausragenden Komplexität der neuronalen Entwicklung und Säugetier-Gehirn-Funktion suchen, die Sensivität dieser hochentwickelten Systemen zu Veränderungen in der Proteinexpression manifestiert sich in einer Reihe von bekannten intellektuellen Defiziten einschließlich Alzheimer und Parkinson-Krankheit (AD und PD) sowie Autismus-Spektrum-Störungen (ASD) wie das Fragile-X-Syndrom (FXS). Letztere Krankheit wird durch eine umfangreiche misexpression einer Vielzahl von Proteinen aus, die auf den Verlust eines einzelnen Übersetzungsregel Protein, FMRP (Fragile X Mental Retardation Protein) 1-4 zurückzuführen ist. Darüber hinaus X - chromosomale Protein A Chromosomenaberrationen , die Variable Ladung zu beeinflussen (VCXA), ein Protein, das mRNA durch Modifizierung der mRNA - Stabilität und Translation verwaltet 5 Capping, haben vor kurzem mit geistigen Defiziten in Verbindung gebracht worden, während Punktmutationen bei Patienten mit kognitiven Behinderungen wurden nicht identifiziert ab sofort 6, 7, was darauf hindeutet , dass die beobachteten psychischen Beeinträchtigungen von veränderten VCXA Ausdruck und dysregulated Ausdruck seiner Ziel Prote stammenins. In Übereinstimmung mit diesen Ergebnissen einer Studie untersucht , ob De - novo - Kopienzahl Variationen von Genen , die mit ASD verbunden sind , festgestellt , dass neuartige Genduplikationen und Streichungen sind ein bedeutender Risikofaktor für ASD 8, also die Idee unterstützen , dass eine erhöhte oder verminderte Proteinexpressionsniveaus führen kann intellektuelle Defizite. Neuere Forschungen weiterer Beweis bereitgestellt bemerkenswert, dass das Expressionsniveau eines gegebenen Proteins genau seine Aggregation als Folge der hohen Proteinmengen nahezu ohne Sicherheitsabstände eingestellt 9 zu verhindern. Es wurde daher vorgeschlagen , dass auch kleine Inkremente ausreichen 9 Krankheiten wie AD und PD zu induzieren. Obwohl die Vielzahl von molekularen Maschinen zur Proteinexpressionskontrolle beiträgt eine komplexe Regelung schlägt Schema in Anbetracht dieser Feststellungen wurde eine Studie zur Untersuchung der Expression von mehr als 5.000 Säugetiergenen 10 Untersuchung gezeigt , dassNatur bevorzugt , ein sparsameres Schema: Die zelluläre Fülle von Proteinen wurde überwiegend auf der Ebene der 10 Übersetzung geregelt gezeigt werden, was zeigt , dass das Management von RNA Verfügbarkeit dient in erster Linie der Proteinexpression zur Feinabstimmung. Untersuchung der Dosis von Proteinen von Interesse (POI), ist daher nicht nur wichtig für das Verständnis der endogene Funktionen eines Proteins, sondern auch auf die Untersuchung vieler Krankheiten und der Entwicklung von Therapien. So haben im vergangenen Jahrzehnten den Fortschritt von mehreren Strategien gesehen RNA-Interferenz mit POI Dosierung zu manipulieren. Obwohl RNA - Interferenz stark Proteinfunktion zu untersuchen verwendet wird und auch in klinischen Studien angewandt wird Krebs oder Augenerkrankungen sowie zu verfolgen antivirale Therapien bei Patienten 11-13, einige Schwierigkeiten auftreten können , zu behandeln , die die Strategie unmöglich machen könnten. Beispielsweise die Samen-Sequenz, die den Zuschlags durch Homologie antreibt, ist comparakurze ble, damit die Förderung der Off-Target-Effekte. Da hocheffiziente Sequenzen selten und müssen unter Tausenden von Optionen ( beschrieben in 14), die Identifizierung der richtigen Reihenfolge gefunden werden kann zeitraubend und kostspielig sein, führt aber immer noch enttäuschend sein kann. Eine alternative Strategie ist, um direkt die POI durch Antikörper zielen. Hier zeigen wir die Verwendung des Proteinträger Chariot (hergestellt von Active Motif) zelluläre Protein Verfügbarkeit zu reduzieren, und der Einsatz von dreidimensionalen Rekonstruktionen folgende Proteinfunktion knock-down zu studieren. Das aktive Motiv des Chariot, selbst ein 2,8 kDa Peptid, wird Shuttle Peptide, Proteine ​​und Antikörper durch Membranen von Säugetierzellen 15 verwendet. Die Peptid assoziiert mit POIs durch Bildung nicht-kovalent gekoppelt makromolekularer Komplexe verwendet hydrophobe Wechselwirkungen, woraufhin Chariot-POI-Komplexe werden in Zellen in einer Endosomen-ind internalisiertependent Weise. Beeinflussen weder die intrazelluläre Lokalisation von Proteinen pendelte wichtiger ist , wurde Chariot zu deutet, noch zytotoxischen Wirkungen auszuüben oder die biologische Aktivität von seiner Ladung 15 zu beeinflussen.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Chariot</td>
			<td>Active Motif</td>
			<td>30025</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life technologies</td>
			<td>21103-049</td>
			<td>warm up to 37&#176;C before using</td>
		</tr>
		<tr>
			<td>1xB27</td>
			<td>Life technologies</td>
			<td>17504044</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Life technologies</td>
			<td>25030-149</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td>&#160;Penicilline and Streptomycine</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td>store at -20&#176;C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td>n.a.</td>
			<td>expensive, but unmatched</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>n.a.</td>
			<td></td>
		</tr>
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studying protein function and the role of altered protein expression by antibody interference and three-dimensional reconstructions

Eine strenge Verwaltung der Proteinexpression ist nicht nur wichtig für jeden Organismus am Leben, sondern auch eine wichtige Strategie Proteinfunktionen in Zellmodellen zu untersuchen. Daher erfunden neuere Forschung verschiedene Werkzeuge Proteinexpression in Säugetierzelllinien oder Tiermodellen, einschließlich RNA und Antikörper Interferenz abzuzielen. Während die erste Strategie viel Aufmerksamkeit in den letzten zwei Jahrzehnten gesammelt hat, Peptide, die eine Translokation von Antikörper Cargos durch Zellmembranen und in Zellen vermitteln, erhalten viel weniger Interesse. In dieser Publikation stellen wir ein detailliertes Protokoll, wie ein Peptidträger namens Chariot in humanen embryonalen Nierenzellen sowie in primären Neuronen im Hippocampus zu nutzen, um Antikörper Interferenzexperimente durchführen und weiter die Anwendung von dreidimensionalen Rekonstruktionen bei der Analyse von Protein-Funktion veranschaulichen. Unsere Ergebnisse legen nahe, dass Chariot ist, wahrscheinlich aufgrund seiner Kernlokalisationssignal, particularly gut geeignet Proteine ​​im soma und den Kern mit Wohnsitz Ziel. Bemerkenswert ist, wenn Chariot zu primären Hippokampus-Kulturen anwenden, wandte sich das Reagenz überraschend gut aus von dissoziierten Neuronen in Kauf genommen werden.

In den folgenden Abschnitten wird eine funktionale Zuschlags eines POI exemplarische Ergebnisse (Simiate, für weitere Details siehe bitte 16, 17) darstellt , unter Verwendung von Chariot Reagenz und Antikörper Interferenz präsentiert werden. Die Ergebnisse zeigen, dass verminderte Expression von Simiate Transkriptionsaktivität beeinträchtigt, und in einer dosisabhängige Weise, induziert Apoptose, die ihren Höhepunkt in Mortalitätsraten von über 99%, wenn höhere Antikörper-Meng...

Watch this Scientific Journal Video about Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions at JoVE.com

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

Das Studium von Proteinfunktionen und die Rolle der Altered Protein Expression von Antikörper-Interferenz und Dreidimensionale Rekonstruktionen

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

studying-protein-function-role-altered-protein-expression-antibody

Research

JoVE Journal

Bioengineering

6.6K Aufrufe.  University of Erlangen-Nuremberg. Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

Serie: Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

Thermodynamik der Membrane Protein Folding durch Fluoreszenz-Spektroskopie

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Forschung

Biotechnik

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Alginat Hydrogele für Three-Dimensional Organ Culture der Eierstöcke und Eileitern

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Planar-und Drei-Dimensional Printing von Leitfähige Druckfarben

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

High-throughput Protein Expression Generator mit einem mikrofluidischen Plattform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Mikrofluidischen Mischern für ein Studium Proteinfaltung

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Untersuchung der dreidimensionalen Strömungstrennung, die durch ein Modell Vocal Fold Polyp induziert wird

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Charakterisierung komplexer Systeme Mit dem Design of Experiments Ansatz: Transient Protein Expression in Tabak als Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Menschliches Knorpelgewebe Herstellung unter Verwendung von dreidimensionalen Inkjet-Drucktechnologie

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Erzeugung eines dreidimensionalen Vollhaut Equivalent und automatisierte Verwundung

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.