Wir sind dankbar für die Ohio State University Comprehensive Cancer Center für die Bereitstellung von Mitteln für diese Arbeit.

1. Cell Line mit integriertem Rekombination Substrate <ol><li> Die HeLa-Zellen wurden stabil mit PDR-GFP (Plasmid 7 Geschenk von M. Jasin, Memorial Sloan-Kettering Cancer Center) umgewandelt und gepflegt in Puromycin für die Auswahl der Transformanten. </li><li> Die Zellen werden in DMEM / FBS passagiert, mit 10% fötalem Rinderserum, 2 mM Glutamax, 1 mM Natriumpyruvat, Penicillin / Streptomycin (100 U / mL/0.1 mg / mL) und 1,5 ug / ml Puromycin. </li></ol> Die Rekombination Substrat in der PDR-GFP-Plasmid, und in stabilen Transformanten, enthält zwei inaktive Allele von GFP (Abbildung 1). Ein Allel ist inaktiv wegen der Aufnahme in deren kodierende Sequenz des 18 bp Restriktionsenzym Erkennungssequenz für I-SceI. Die Expression von I-SceI in diesen Zellen erzeugt eine doppelsträngige DNA (DSB). Es gibt eine zweite Allel des inaktiven GFP auf dieser DNA, sondern den Teil seiner Sequenz mit der I-SceI auf dem ersten Allel korreliert ist Wildtyp. Diese zweite GFP-Allels kann als eine Folge der Geber für die homologe Rekombination Reparatur der DSB-Funktion und würde in eine aktive GFP-Allels, die sich leicht mittels Durchflusszytometrie detektiert führen. Alternativ könnte der DSB durch homologe End-Verbindung, welche die I-SceI Restriktionsstelle zerstören würde und in GFP-negativen Zellen führen repariert werden. Die Höhe der homologen Rekombination in einer Zelle wird durch das Zählen der Anteil der GFP-positiven Zellen bestimmt. Diese Zelllinie, pHeLa-DR13-9, wurde bei allen Klonen für Null-Hintergrund GFP-Signal und ein relativ hoher Prozentsatz von GFP-positive Zellen nach Transfektion mit dem I-SceI exprimierenden Plasmid, pCBASce ausgewählt. (Die pCBASce und Vektor-Regelung, pCAGGS, wurden beide von M. Jasin des Memorial Sloan-Kettering Cancer Center geliefert.) Die HeLa-DR13-9-Zelllinie ist von den Autoren auf Anfrage erhältlich. 2. Transfektion zu einem Abbau endogener Gene BRCA1 und Add Zurück Mutant BRCA1. <ol><li> Am Tag vor der ersten Transfektion (in der Regel Dienstag): mit einer 10 cm Schüssel mit HeLa-DR13-9-Zellen mindestens 90% konfluent Begin. </li><li> Trypsinize in 1 mL und stoppen Sie die Reaktion durch Zugabe von 9 ml DMEM / FBS, gut mischen durch Pipettieren. </li><li> Add 40 ul trypsiniert Zellen pro Vertiefung einer 24-Well-Platte in 0,5 ml DME / FBS. </li><li> Tag 1 Transfektionen (Mittwoch): Zellen sollten ~ 40-50% konfluent sein, wenn viel weniger anfangen. Transfizieren durch Mischen: 5 pmol siRNA + 0,3 ug BRCA1-Mutanten exprimierenden Plasmid DNA in 12,5 ul Opti-MEM und 0,5 ul Lipofectamine 2000 in 12,5 ul Opti-MEM. Fahren Sie mit der Transfektion nach dem Lipofectamine Protokoll (Invitrogen). </li><li> Ersetzen Sie die Medien 4-6 Stunden später. Die siRNA wir nutzen, um BRCA1 führen auf die Reihenfolge GCUCCUCUCACUCUUCAGU Angabe BRCA1 3&#39;-UTR-Sequenzen 80.780 bis 80.798 (Zugangsnummer AY273801) basiert. </li><li> Tag 2: Transfer der Zellen durch trypsinizing von der 24-Well-Platte zu einer 6-Well-Platte. </li><li> Tag 3 Transfektionen: 25 pmol siRNA + 0,75 ug BRCA1-Mutanten exprimierenden Plasmid DNA + 0,75 ug pCBASce (oder pCAGGS vector control) in 62,5 ul Opti-MEM und 2,5 ul Lipofectamine 2000 in 62,5 ul Opti-MEM. Ersetzen Sie die Medien 4-6 Stunden später. </li></ol> 3. Die Analyse der Transfektanten. <ol><li> Am Tag 6, trypsinize Zellen aus jeder Vertiefung und stoppen in 1 ml DMEM / FBS. Analysieren der Zellen für die GFP-Expression kann an den Tagen 5 oder 7 gemacht werden, aber wir finden, dass Tag 6 am besten funktioniert. </li><li> Analysieren 10.000 Zellen pro Vertiefung mittels Durchflusszytometrie. Wir verwenden eine Becton Dickinson FACSCalibur Instrument in der Ohio State University Comprehensive Cancer Center Analytical Cytometry freigegebenen Ressource. Single, lebende Zellen sind gated mit der Vorwärts-und Seitwärts-Streulicht-Parameter. GFP-positiven Zellen werden unter Verwendung der GFP-Fluoreszenz-Detektor (FL1), und die Ergebnisse werden als Histogramm (Abbildung 2, unten) dargestellt. </li><li> Übrig Zellen können für die Fotografie durch Fluoreszenzmikroskopie replattiert werden, falls gewünscht. </li></ol> 4. Repräsentative Ergebnisse: BRCA1 regelt den Weg für die Reparatur von doppelsträngigen DNA-Brüchen durch homologe Rekombination 6,11. Jene Zellen, die Reparatur unterzogen haben, durch homologe Rekombination werden leicht durch Fluoreszenzmikroskopie (Abbildung 2, oben) nachgewiesen. Depletion von BRCA1 führt zu einer Abnahme der detektierten GFP-positiven Zellen (Abbildung 2, rechts). Die Ausgabe aus dem FACSCalibur ist ein Histogramm mit GFP-Intensität pro Zelle auf der X-Achse und die Anzahl der Zellen auf der Y-Achse (Abbildung 2, unten). In einem typischen Satz von Ergebnissen aus einem einzigen Experiment wurden 15,5% der Zellen GFP-positive nach I-SceI Ausdruck und Kontrolle RNAi Depletion (Abbildung 3). Zum Vergleich: add Erschöpfung der BRCA1-und Vektor zurück GFP-Umstellung auf 0,7% reduziert. Add-back von Wildtyp-BRCA1 oder BRCA1 (I21V) vollständig wiederhergestellt homologe Rekombination, wie durch den Erwerb 15-16% GFP-positive Zellen nachgewiesen. Add-back des BRCA1 (M18T)-Mutante (Abbildung 3, Spur 5) hatte ähnliche Wirkung wie das Add-back des Vektor-Regelung. Da diese Variante auf ähnlichem Niveau wie das endogene Protein 9 ausgedrückt, wir so interpretieren die BRCA1 (M18T) Variante ist nicht funktionsfähig in homologe Rekombination. Wir haben in der Regel 10-20% der Zellen zu konvertieren GFP-positive nach Transfektion der I-SceI Expressionsplasmid. Depletion von BRCA1 reproduzierbar reduziert die Umwandlung von GFP-positiven Zellen um etwa 8-10 fache. Obwohl der tatsächliche Anteil der GFP-positive Zellen können von Experiment zu Experiment, das Verhältnis von GFP-positive Zellen nach BRCA1 Abbau bezogen auf die Kontrolle Abbau innerhalb eines Experiments zu ändern ist ziemlich konstant. Wir normieren Ergebnisse, indem Sie die ungehemmte Umrechnungskurs zu 100% und Express-Ergebnisse mit BRCA1 Abreicherungen und Hinzurechnungen in Prozent. Auf diese Weise können wir kombinieren mehrere Experimente, und die experimentelle Variation ist nicht hoch. Add Rückseite des BRCA1-Mutanten haben bisher ergeben robuste Ergebnisse: jeweils BRCA1-Variante hat entweder vollständig homologe Rekombination Aktivität wieder hergestellt hat oder hatte keine Wirkung. Eine wichtige Überlegung bei der Erlangung dieser Ergebnisse ist, dass unsere Transfektionsbedingungen Ebenen des BRCA1-Protein, das etwa gleich der Konzentration des endogenen Proteins in unbehandelten Zellen zu produzieren. Zu hohe einer Überexpression von selbst eine defekte Mutante von BRCA1 in manchen positive homologe Rekombination Aktivität führen. Ein Problem, das wir zu überwinden brauchte, war die Toxizität der Transfektion. Depletion von BRCA1 selbst kann langsam das Zellwachstum, und das Gleichgewicht der Lipofectamine-2000 und Plasmid-DNA bezogen auf die Anzahl der Zellen auf der Schale muss ein einheitliches Niveau gehalten werden. Zu viel Lipofectamine oder Plasmid-DNA reduzieren Zellzahlen und die Anzahl der Zellen nicht ausreichen werden, um zuverlässige Durchflusszytometrie Ergebnisse zu erhalten. Der Saldo aus dem Plasmid und Lipofectamine Reagenzien im Vergleich zu der Anzahl der Zellen transfiziert ist sehr wichtig für die Aufrechterhaltung der Lebensfähigkeit der Zellen. <img src="/files/ftp_upload/2468/2468fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Die Rekombination Substrat. Die Strategie, die von der Gruppe von M. Jasin 7,12 entwickelt worden war dargestellt. Die PDR-GFP-Plasmid enthält zwei defekte Allele von GFP, von denen ein I-SceI Restriktionsendonukleasestelle enthält. Ein HeLa abgeleitete Zelllinie enthält dieses DNA-Substrat an einem Ort im Genom 9 integriert und aktiv die Reparatur der doppelsträngigen DNA-Pause auf der I-SceI Ort durch homologe Rekombination Ergebnisse bei der Umwandlung von einem Allel zu GFP-positiv. <img src="/files/ftp_upload/2468/2468fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Erkennung von GFP-positiven Zellen durch Fluoreszenzmikroskopie. Die Zellen wurden nach diesem Protokoll getestet und Kontrolle verarmten Zellen wurden in der homologen Rekombination aktiv, wie durch einen hohen Anteil von Zellen mit GFP-Expression (links) erkannt. Depletion von BRCA1 durch RNAi führt zu einem starken Rückgang in der Zahl der GFP-positiven Zellen (rechts). <img src="/files/ftp_upload/2468/2468fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Ergebnisse einer Hinzurechnung von BRCA1 Missense-Mutanten. Ergebnisse aus einem einzigen Experiment sind in diesem Histogramm angezeigt. In Abwesenheit von transfizierten I-SceI exprimierenden Plasmid (Spur 1) gibt es keine GFP-positive Zellen nachgewiesen. Die Ergebnisse in den Spuren 2-6 wurden alle aus Zellen, in denen die I-SceI exprimierenden Plasmid am Tag 3 transfiziert wurde. In Zellen, die für eine irrelevante Gen (Luciferase) aufgebraucht waren und mit Vektor-add-back, waren 15,5% der Zellen GFP-positive (Spur 2). Depletion von BRCA1-und Vektor-add-back zeigt die Wirkung der Verlust von BRCA1 auf homologe Rekombination (Spur 3). Die Auswirkungen der Verarmung der Gene BRCA1 und Hinzurechnung von Wildtyp-BRCA1 (Bahn 4), BRCA1 (M18T) (Spur 5) und BRCA1 (I21V) (Spur 6) werden angezeigt. Diese Ergebnisse stammen aus einem Experiment, und würde mit mindestens zwei weitere Experimente zu wiederholen kombiniert werden, um ein Maß für die homologe Rekombination von einem mutierten BRCA1-Protein unterstützt zu erhalten.

<ol>
	<li>Carvalho, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+cancer+risk+associated+with+germ+line+BRCA1+missense+variants+by+functional+analysis.">Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis.</a> Cancer Res. 67, 1494-1501 (2007).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+epidemiology+of+BRCA1.">Genetic epidemiology of BRCA1.</a> Cancer Biol Ther. 3, 509-514 (2004).</li><li>Couch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+functional+effects+of+unclassified+genetic+variants.">Assessment of functional effects of unclassified genetic variants.</a> Hum Mutat. 29, 1314-1326 (2008).</li><li>Tomlinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+breast+cancer+cell+line+derived+from+a+germ-line+BRCA1+mutation+carrier.">Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier.</a> Cancer Res. 58, 3237-3242 (1998).</li><li>Ruffner, H., Joazeiro, C. A., Hemmati, D., Hunter, T., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-predisposing+mutations+within+the+RING+domain+of+BRCA1:+loss+of+ubiquitin+protein+ligase+activity+and+protection+from+radiation+hypersensitivity.">Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity.</a> Proc Natl Acad Sci U S A. 98, 5134-5139 (2001).</li><li>Moynahan, M. E., Chiu, J. W., Koller, B. H., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brca1+controls+homology-directed+DNA+repair.">Brca1 controls homology-directed DNA repair.</a> Mol Cell. 4, 511-518 (1999).</li><li>Nakanishi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Fanconi+anemia+monoubiquitination+pathway+promotes+homologous+DNA+repair.">Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.</a> Proc Natl Acad Sci U S A. 102, 1110-1115 (2005).</li><li>Pierce, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+breaks+and+tumorigenesis.">Double-strand breaks and tumorigenesis.</a> Trends Cell Biol. 11, 52-59 (2001).</li><li>Ransburgh, D. J., Chiba, N., Ishioka, C., Toland, A. E., Parvin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+breast+tumor+mutations+in+BRCA1+that+abolish+its+function+in+homologous+DNA+recombination.">Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination.</a> Cancer Res. 70, 988-995 (2010).</li><li>Easton, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+genetic+assessment+of+1,433+sequence+variants+of+unknown+clinical+significance+in+the+BRCA1+and+BRCA2+breast+cancer-predisposition+genes.">A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes.</a> Am J Hum Genet. 81, 873-883 (2007).</li><li>Snouwaert, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1+deficient+embryonic+stem+cells+display+a+decreased+homologous+recombination+frequency+and+an+increased+frequency+of+non-homologous+recombination+that+is+corrected+by+expression+of+a+brca1+transgene.">BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a brca1 transgene.</a> Oncogene. 18, 7900-7907 (1999).</li><li>Pierce, A. J., Hu, P., Han, M., Ellis, N., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ku+DNA+end-binding+protein+modulates+homologous+repair+of+double-strand+breaks+in+mammalian+cells.">Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells.</a> Genes Dev. 15, 3237-3242 (2001).</li><li>Spearman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinically+applicable+models+to+characterize+BRCA1+and+BRCA2+variants+of+uncertain+significance.">Clinically applicable models to characterize BRCA1 and BRCA2 variants of uncertain significance.</a> J Clin Oncol. 26, 5393-5400 (2008).</li><li>Sweet, K., Senter, L., Pilarski, R., Wei, L., Toland, A. 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Der Vorteil dieser Methode ist, dass wir die Funktion des BRCA1-Protein bei der Regulation der DNA-Reparatur durch homologe Rekombination unter Verwendung von Zellen, die Wildtyp-endogen exprimierten Gene BRCA1 haben zu studieren. Wir verfügen über zwei separate etablierten Methoden der Untersuchung der homologen Rekombination und der Nutzung RNAi-vermittelten Abbau der neuartigen Methode zum Testen von BRCA1-Varianten für die Funktion der DNA-Reparatur erhalten hat. Der Schlüssel für den Erfolg dieser Methode war es, eine leicht transfizierbare Zelllinie, wie HeLa etablieren, mit der Rekombination Substrat in das Genom, so dass wir sehr hohe GFP Umsatz zu erreichen. Wir hatten mehrere Klone des ursprünglichen Transformation, um gesiebt, um eine, die keine nachweisbaren Hintergrund GFP-Signal war zu erhalten, sondern auf die Transfektion der I-SceI Expressionsplasmid hatte ein sehr hohes Maß an GFP-Konvertierung. Mit dieser Methode sind wir nun untersuchen andere spezifische BRCA1 Aminosäurereste für die Funktion in der homologen Rekombination. Zusammen mit der biologischen Erkenntnisse aus dieser Arbeit lassen sich diese Schlussfolgerungen für die klinische Krebsgenetik angewendet werden. Ein hoher Prozentsatz der BRCA1 Missense-Mutationen haben unbekannte klinische Bedeutung, da es viele seltene Varianten und es gibt nicht genügend Informationen für viele dieser, um den Krebs Verein durch genetische Segregationsanalyse 3,10,13,14 zu bestimmen. Mit dieser Methode könnten die Folgen einer spezifischen Variante der biologischen Funktion von BRCA1 bei der Regulierung der homologen Rekombination zur Frauen, die eine dieser Varianten tragen in ihrem Genom zu informieren.

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identifying the effects of brca1 mutations on homologous recombination using cells that express endogenous wild-type brca1

Die funktionelle Analyse von Missense-Mutationen können durch die Anwesenheit in der Zelle des endogenen Proteins kompliziert sein. Struktur-Funktions-Analysen des BRCA1 haben durch das Fehlen einer robusten Assay für die volle Länge BRCA1-Protein und die Schwierigkeiten in der Arbeit mit Zelllinien, die hypomorphen BRCA1-Protein 1,2,3,4,5 äußern kompliziert. Wir entwickelten ein System, bei dem endogenen BRCA1-Protein in einer Zelle akut durch RNAi-Targeting der 3&#39;-UTR des BRCA1 mRNA erschöpft war und durch Co-Transfektion eines Plasmids, das eine BRCA1-Variante. Ein Vorteil dieses Verfahrens ist, dass die akute Schweigen der Gene BRCA1 und gleichzeitigen Austausch der Zellen ohne sekundäre Mutationen oder Anpassungen, die im Laufe der Zeit entstehen könnten, um den Verlust von BRCA1-Funktion kompensieren wachsen lassen. Dieser Abbau und Add-back-Verfahren wurde in einem HeLa-derived-Zelllinie, die leicht für die homologe Rekombination Aktivität wurde untersucht getan. Die homologe Rekombination Test basiert auf einem bereits veröffentlichten Methode, bei der eine Rekombination Substrat in das Genom (Abbildung 1) 6,7,8,9 integriert ist. Diese Rekombination Substrat hat die seltene-Schneid I-SceI Restriktionsenzymstelle in einem inaktiven GFP-Allel und nachgeschaltet ist eine zweite inaktive GFP-Allel. Transfektion der Plasmid, das I-SceI Ergebnisse drückt in einem doppelsträngigen Pause, die durch homologe Rekombination repariert werden kann, und wenn homologe Rekombination nicht reparieren der Pause schafft eine aktive GFP-Allel, das ist leicht mittels Durchflusszytometrie für GFP-Protein-Expression erzielt . Depletion von endogenen BRCA1 führte zu einer 8-10-fache Reduktion der homologen Rekombination Aktivität und Hinzurechnung von Wildtyp-Plasmid vollständig restauriert homologe Rekombination Funktion. Wenn bestimmte Punktmutanten in voller Länge BRCA1 von co-transfiziert Plasmiden exprimiert wurden, konnte die Wirkung des spezifischen Missense-Mutanten erzielt werden. Als ein Beispiel, der Ausdruck des BRCA1 (M18T)-Protein, eine Variante des unbekannten klinische Bedeutung 10, in diesen Zellen exprimiert wurde, unterließ es BRCA1-abhängigen homologen Rekombination wiederherstellen. Im Gegensatz dazu Ausdruck einer anderen Variante auch von unbekannter Bedeutung, BRCA1 (I21V) voll BRCA1-abhängigen homologen Rekombination Funktion wiederhergestellt. Diese Strategie der Prüfung der Funktion des BRCA1 Missense-Mutationen wurde in einen anderen biologischen System Testen auf Zentrosomenfunktion (Kais et al, unveröffentlichte Beobachtungen) angewendet worden. Insgesamt ist dieser Ansatz für die Analyse von Missense-Mutanten in einem Gen, das rezessiv analysiert werden müssen.

Watch this Scientific Journal Video about Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1 at JoVE.com

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

Wir stellen eine Methode zur Prüfung von BRCA1-Varianten in einer Gewebekultur basierter Assay für die homologe Rekombination Reparatur von DNA-Schäden, die durch abbauende endogenen BRCA1-Protein aus einer Zelle mit RNAi und ersetzt sie mit einer BRCA1 Punktmutante, dass eine Codierung ändern enthält.

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 ...

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14.6K Aufrufe.  The Ohio State University. Wir stellen eine Methode zur Prüfung von BRCA1-Varianten in einer Gewebekultur basierter Assay für die homologe Rekombination Reparatur von DNA-Schäden, die durch abbauende endogenen BRCA1-Protein aus einer Zelle mit RNAi und ersetzt sie mit einer BRCA1 Punktmutante, dass eine Codierung ändern enthält.

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

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Untersuchung der Auswirkungen von Matrix Stiffness auf Cellular-Funktion mit Acrylamid-basierte Hydrogele

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Forschung

Biologie

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Die Analyse der Funktion von kleinen GTPasen durch Mikroinjektion von Plasmiden in polarisierten Epithelzellen

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Ein PCR-basierte Genotypisierung Methode, zwischen Wildtyp-und Ziersorten Unterscheiden von Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombineering homologen Rekombination Konstrukte in<em&gt; Drosophila</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Messung der Auswirkungen von Bakterien auf<em&gt; C. Elegans</em&gt; Verhalten Mit einem Ei Retention Assay

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

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 ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Ermittlung von Protein-Protein-Interaktionsstellen unter Verwendung von Peptid-Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Bestimmung von Sialinsäuren in Leber- und Milchproben von Wildtyp und<em&gt; CMAH</em&gt; Knock-out Mäuse.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Eine Methode zur Beurteilung von Bakteriocin-Wirkungen auf die Gut-Mikrobiota von Mäusen

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Untersucht die nachteiligen Effekte der Low Druck Plasmasterilisation auf das Überleben von Bacillus Subtilis Sporen mittels Live Cell Mikroskopie

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Methods Article

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