Name: analysis of dna double-strand break (dsb) repair in mammalian cells
Uploaded: 2010-09-08T17:00:00
Description: Watch this Scientific Journal Video about Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells at JoVE.com

De originele GFP-Pem1 was een geschenk van Dr Lei Li. Dit werk werd ondersteund door subsidies van de NIH en de Ellison Medical Foundation om VG en AS

In dit protocol beschrijven we de methode voor de analyse van DNA DSB repareren met chromosomaal geïntegreerde reporter constructen 1,2 waar de DSB&#39;s zijn in vivo veroorzaakt door transiënte expressie een zeldzame cutting endonuclease I-SCEI 3. De geïntegreerde test biedt het voordeel van het analyseren van DSB repareren in de chromosomale context. Echter, dit protocol vereist langdurige cel passage, die problematisch kan zijn bij het werken met primaire cellen. <p class="jove_c...

<ol>
	<li>Mao, Z., Seluanov, A., Jiang, Y., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRF2+is+required+for+repair+of+nontelomeric+DNA+double-strand+breaks+by+homologous+recombination.">TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination.</a> Proc Natl Acad Sci U S A. 104, 13068-13073 (2007).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+nonhomologous+end+joining+and+homologous+recombination+in+human+cells.">Comparison of nonhomologous end joining and homologous recombination in human cells.</a> DNA Repair (Amst). 7, 1765-1771 (2008).</li><li>Rouet, P., Smih, F., 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=Expression+of+a+site-specific+endonuclease+stimulates+homologous+recombination+in+mammalian+cells.">Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.</a> Proc Natl Acad Sci U S A. 91, 6064-6068 (1994).</li><li>Mao, Z., Jiang, Y., Xiang, L., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+homologous+recombination,+but+not+by+nonhomologous+end+joining,+is+elevated+in+breast+cancer+cells.">DNA repair by homologous recombination, but not by nonhomologous end joining, is elevated in breast cancer cells.</a> Neoplasia. 11, 683-691 (2009).</li><li>Seluanov, A., Mittelman, D., Pereira-Smith, O. M., Wilson, J. H., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+joining+becomes+less+efficient+and+more+error-prone+during+cellular+senescence.">DNA end joining becomes less efficient and more error-prone during cellular senescence.</a> Proc Natl Acad Sci U S A. 101, 7624-7629 (2004).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+nonhomologous+end+joining+and+homologous+recombination+during+cell+cycle+in+human+cells.">DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.</a> Cell Cycle. 7, 2902-296 (2008).</li></ol>

De fluorescerende NHEJ en HR-reporter testen leveren een kwantitatieve manier voor het meten van elk afzonderlijk DSB reparatie pathway in vivo. De testen zijn zeer gevoelig, omdat FACS op betrouwbare wijze kan 10 GFP + cellen te detecteren in 20.000 cellen. De assays kan worden aangepast voor het meten van herstel gebeurtenissen in &quot;real time&quot; door het detecteren van de verschijning van GFP + cellen binnen enkele minuten of uren na inductie van DSB&#39;s 2. Bovendien is de analyse van de G...

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<td>EndoFree Plasmid Maxi kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12362</td>
<td>&nbsp;</td>
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<td>Qiaex II Gel Extraction Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>20021</td>
<td>&nbsp;</td>
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<td>Amaxa Nucleofector</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>AAD-1001</td>
<td>&nbsp;</td>
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<td>Geneticin (G418)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11811-031</td>
<td>&nbsp;</td>
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<td>pDsRed2-N1</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>632406</td>
<td>&nbsp;</td>
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<td>Round bottom tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352058</td>
<td>FACS tubes</td>
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analysis of dna double-strand break (dsb) repair in mammalian cells

DNA dubbelstrengs breuken zijn de meest gevaarlijke DNA laesies die kunnen leiden tot massale verlies van genetische informatie en celdood. Cellen repareren DSB&#39;s met behulp van twee belangrijke routes: niet-homologe eind mee (NHEJ) en homologe recombinatie (HR). Verstoringen van de NHEJ en HR worden vaak geassocieerd met vroegtijdige veroudering en het ontstaan ​​van tumoren, daarom is het belangrijk om een ​​kwantitatieve manier van meten elk DSB reparatie pad. Ons laboratorium heeft ontwikkeld fluorescerende reporter constructen die gevoelige en kwantitatieve meting van NHEJ en HR mogelijk te maken. De constructen zijn gebaseerd op een aangelegde GFP-gen bevat erkenning sites voor een zeldzame-cutting I-SCEI endonuclease voor inductie van DSB. Het uitgangspunt GFP constructen zijn negatief omdat het GFP-gen wordt geïnactiveerd door een extra exon, of door mutaties. Succesvolle reparatie van de I-SCEI-geïnduceerde pauze door NHEJ of HR herstelt het functionele GFP-gen. Het aantal GFP positieve cellen geteld door flowcytometrie geeft kwantitatieve meting van NHEJ of HR-efficiëntie.

Watch this Scientific Journal Video about Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells at JoVE.com

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

Dit artikel beschrijft op basis van GFP-fluorescentie<em&gt; In vivo</em&gt; Testen die homologe recombinatie afzonderlijk te kwantificeren en niet-homologe eind mee in zoogdiercellen.

Analyse van DNA dubbelstrengs breuken (DSB) Reparatie in zoogdiercellen

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

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Biology

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

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