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><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>&amp;nbsp;D-001810-10-05</td><td>ON-TARGETplus Non-targeting Pool</td></tr><tr><td>Lipofectamine RNAiMAX Reagent&amp;nbsp;</td><td>Life Technologies</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/13778075&quot;&gt;13778075&lt;/a&gt;</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&amp;nbsp;</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&amp;nbsp; 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>1:1,000 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

Universidad de Cartagena UDEC

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

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

Functional Genomics Screening Verwendung zur Identifizierung möglicher neuer Wirkstoff-Targets in Cancer Cell Sphäroid-Kulturen

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Forschung

Krebsforschung

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C&#62;T, c.154C&#62;T, c.3847C&#62;T, c.5056C&#62;T, and c.4986+5G&#62;A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

Funktionsbewertung von BRCA1-Varianten mit CRISPR-mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Isolierung und funktionelle Beurteilung menschlicher Brustkrebsstammzellen aus Zell- und Gewebeproben

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Profiling von Östrogen-regulierte MicroRNAs in Brustkrebszellen

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Medizin

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Bewertung der Wirksamkeit der Krebsmedikament Sensibilisierung<em&gt; In-vitro-</em&gt; Und<em&gt; In-vivo-</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

Ermittlung der Auswirkungen von BRCA1-Mutationen auf homologe Rekombination unter Verwendung von Zellen, die endogenes Wildtyp-BRCA1 Express

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Biologie

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

In vitro Auswahl von modifizierten Transkriptionsrepressoren für gezieltes epigenetisches Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Biotechnik

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 ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

gDNA Enrichment durch eine Transposase-basierte Technologie für NGS-Analyse der gesamten Sequenz von<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt; Und 9 Gene in der DNA-Reparatur beteiligt Schäden

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Silencing

Zusammenfassung

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