Name: two- and three-dimensional live cell imaging of dna damage response proteins
Uploaded: 2012-09-28T12:00:00
Description: Watch this Scientific Journal Video about Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins at JoVE.com

R01NS064593 और R21ES016636 (केवी) के हिस्से में समर्थन. माइक्रोस्कोपी VCU पर प्रदर्शन किया गया था - तंत्रिका जीव विज्ञान और एनाटॉमी माइक्रोस्कोपी सुविधा का विभाग, NIH NINDS केंद्र कोर अनुदान 5P30NS047463 से धन के साथ भाग में, समर्थित. कताई डिस्क confocal खुर्दबीन एक पुरस्कार NIH-NCRR साथ (1S10RR027957) खरीदा गया था.

ए सेल तैयारी <ol><li> सामान्य मानव प्राथमिक fibroblasts (GM02270) Coriell सेल भंडार, Camden, न्यू जर्सी, से प्राप्त किया गया और 6 hTERT साथ अमर. कोशिकाओं बड़े हो रहे थे और मीडिया में Cellstar में विस्तार 6 सेमी व्यंजन (4 मिलीलीटर) 20% भ्रूण ग?...

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+to+study+DNA+repair:+what's+in+the+box.">Tools to study DNA repair: what's in the box.</a> Trends Genet. 24, 467-474 (2008).</li><li>Giunta, S., Belotserkovskaya, R., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+damage+signaling+in+response+to+double-strand+breaks+during+mitosis.">DNA damage signaling in response to double-strand breaks during mitosis.</a> J. Cell Biol. 190, 197-207 (2010).</li><li>Giunta, S., Jackson, S. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone-GFP+fusion+protein+enables+sensitive+analysis+of+chromosome+dynamics+in+living+mammalian+cells.">Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells.</a> Curr. Biol. 8, 377-385 (1998).</li><li>Massignani, M., Canton, I., Sun, T., Hearnden, V., Macneil, S., Blanazs, A., Armes, S. P., Lewis, A., Battaglia, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+fluorescence+imaging+of+live+cells+by+effective+cytosolic+delivery+of+probes.">Enhanced fluorescence imaging of live cells by effective cytosolic delivery of probes.</a> PLoS One. 5, e10459 (2010).</li><li>Nakamura, K., Sakai, W., Kawamoto, T., Bree, R. T., Lowndes, N. F., Takeda, S., Taniguchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+vertebrate+53BP1:+a+major+role+in+non-homologous+end+joining+of+DNA+double+strand+breaks.">Genetic dissection of vertebrate 53BP1: a major role in non-homologous end joining of DNA double strand breaks.</a> DNA Repair (Amst). 5, 741-749 (2006).</li><li>Schultz, L. B., Chehab, N. H., Malikzay, A., Halazonetis, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+binding+protein+1+(53BP1)+is+an+early+participant+in+the+cellular+response+to+DNA+double-strand+breaks.">p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks.</a> J. Cell. Biol. 151, 1381-1390 (2000).</li><li>Valerie, K., Povirk, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+mechanisms+of+mammalian+double-strand+break+repair.">Regulation and mechanisms of mammalian double-strand break repair.</a> Oncogene. 22, 5792-5812 (2003).</li><li>Wang, B., Matsuoka, S., Carpenter, P. B., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1,+a+mediator+of+the+DNA+damage+checkpoint.">53BP1, a mediator of the DNA damage checkpoint.</a> Science. 298, 1435-1438 (2002).</li><li>Ward, I. M., Minn, K., Jorda, K. 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जीनोमिक अखंडता के रखरखाव सेल अस्तित्व के लिए महत्वपूर्ण है. समय से पहले बूढ़ा, carcinogenesis, या 8 मौत में जीनोम परिणाम के संरक्षण में विफलता. समझदार में गहन रुचि है कैसे DDR कार्यों, दोनों बुनियादी और नैदानिक ?...

<table border="1"><tbody><tr><td> उत्पाद </td><td> कंपनी </td></tr><tr><td> Cellstar संस्कृति व्यंजन </td><td> जैव एक Greiner </td></tr><tr><td> FluroDish गिलास नीचे व्यंजन </td><td> विश्व प्रेसिजन उपकरण इंक, </td></tr><tr><td> सदस्य मीडिया </td><td> GIBCO </td></tr><tr><td> गैर आवश्यक अमीनो एसिड </td><td> GIBCO </td></tr><tr><td> एमिनो एसिड </td><td> GIBCO </td></tr><tr><td> विटामिन </td><td> GIBCO </td></tr><tr><td> सोडियम पाइरूवेट </td><td> Invitrogen </td></tr><tr><td> पेनिसिलिन स्ट्रेप्टोमाइसिन / </td><td> HyClone </td></tr><tr><td> भ्रूण गोजातीय सीरम </td><td> GIBCO </td></tr><tr><td> N-Myc - 53BP1 WT pLPC Puro; 19836 प्लाज्मिड </td><td> Addgene </td></tr><tr><td> pCLNR H2BG, 17735 प्लाज्मिड </td><td> Addgene </td></tr><tr><td> SuperFect </td><td> क्विएज़न </td></tr><tr><td> Zeiss सेल प्रेक्षक एसडी इमेजिंग प्रणाली </td><td> Zeiss </td></tr><tr><td> AxioVision (4.8.2 रिहाई) </td><td> Zeiss </td></tr><tr><td> Zeiss Immersol डब्ल्यू तेल </td><td> Zeiss </td></tr><tr><td> Volocity सॉफ्टवेयर (संस्करण 6.0) </td><td> PerkinElmer </td></tr></tbody></table>

two- and three-dimensional live cell imaging of dna damage response proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

Watch this Scientific Journal Video about Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins at JoVE.com

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

इस प्रोटोकॉल एक डीएनए डबल कतरा तोड़ संकेतन प्रोटीन डीएनए के रूप में के रूप में अच्छी तरह से पिंजरे का बँटवारा दौरान अपने स्थानीयकरण क्षति प्रतिक्रिया में सक्रिय visualizing के लिए एक विधि का वर्णन है.

दो और तीन आयामी डीएनए क्षति प्रतिक्रिया प्रोटीन की लाइव सेल इमेजिंग

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

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