Name: study of the dna damage checkpoint using xenopus egg extracts
Uploaded: 2012-11-05T13:00:00
Description: Watch this Scientific Journal Video about Study of the DNA Damage Checkpoint using Xenopus Egg Extracts at JoVE.com

Dette arbejde er delvist understøttet af midler fra The University of North Carolina i Charlotte, Wachovia fundament fond til fakultet ekspertise, og et tilskud fra NIGMS (R15GM101571).

1. LSE Forberedelse <ol><li> Kvindelige frøer (Xenopus laevis) injiceres to gange for ægudtagning. Den første injektion (priming) er 100 U PMSG (Gravid Mare Serum Gonadotropin) per frø. Frøer primes mindst to dage før induktion æglægning og primede frøer kan anvendes i op til to uger. Til premierminister frøer, PMSG subkutant i de dorsale lymfeknuder sacs anvendelse af en 3 ml sprøjte og 27 G nål injiceres. </li><li> For at inducere æglægning, injiceres 500 U hCG (humant choriongonadotropin) pr ...

<ol>
	<li>Lohka, M. J., Masui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+in+vitro+of+sperm+pronuclei+and+mitotic+chromosomes+induced+by+amphibian+ooplasmic+components.">Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.</a> Science. 220 (4598), 719-721 (1983).</li><li>Cimprich, K. A., Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATR:+an+essential+regulator+of+genome+integrity.">ATR: an essential regulator of genome integrity.</a> Nat. Rev. Mol. Cell Biol. 9 (8), 616-627 (2008).</li><li>Lupardus, P. J., Van, C., Cimprich, K. 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E., Costanzo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mre11-Rad50-Nbs1-dependent+processing+of+DNA+breaks+generates+oligonucleotides+that+stimulate+ATM+activity.">Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity.</a> EMBO. J. 27 (14), 1953-1962 (2008).</li><li>Kumagai, A., Dunphy, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Claspin,+a+novel+protein+required+for+the+activation+of+Chk1+during+a+DNA+replication+checkpoint+response+in+Xenopus+egg+extracts.">Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts.</a> Mol. Cell. 6 (4), 839-849 (2000).</li><li>Yan, S., Lindsay, H. D., Michael, W. 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Der er flere fordele ved at studere DNA beskadigelse checkpoint ved hjælp af Xenopus æg ekstrakter. Anvendelsen af ​​æg ekstrakter giver en stor mængde cellefri ekstrakter synkroniseret ved interfasen af ​​cellecyklussen. Ægget ekstrakter kan let og billigt fremstillet. Det er relativt let at beskadige DNA eller kromatin og at afsløre en fejl i DNA beskadigelse checkpoint&#39;et efter immunodepleting et målprotein fra æggeekstrakt. Efterfølgende kan en potentiel funktion defekt blive &quot;redd...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Reagenser </td></tr><tr><td> Anti-Chk1 P-S344-antistof </td><td> Cellesignalering </td><td> 2348L </td><td></td></tr><tr><td> Anti-Chk1 antistof </td><td> Santa Cruz </td><td> SC7898 </td><td></td></tr><tr><td> Aprotinin </td><td> MP Biomedicals </td><td> 0219115880 </td><td></td></tr><tr><td> Cycloheximid </td><td> Sigma </td><td> C7698-5G </td><td></td></tr><tr><td> Cytochalasin B </td><td> EMD </td><td> 250.233 </td><td></td></tr><tr><td> Dithiothreitol (DTT) </td><td> VWR </td><td> JTF780-2 </td><td></td></tr><tr><td> hCG </td><td> Sigma </td><td> CG10-10VL </td><td></td></tr><tr><td> L-cystein </td><td> Sigma </td><td> C7352-1KG </td><td></td></tr><tr><td> Leupeptin </td><td> VWR </td><td>97063-922 </td><td></td></tr><tr><td> Methylmethansulfonat (MMS) </td><td> Sigma </td><td> 129925-5G </td><td></td></tr><tr><td> Nocodazol </td><td> Sigma </td><td> M1404-2mg </td><td></td></tr><tr><td> PMSG </td><td> Calbiochem </td><td> 367.222 </td><td></td></tr><tr><td> Prøvepuffer </td><td> Sigma </td><td> S3401 </td><td></td></tr><tr><td> Tautomycin </td><td> Wako Chemicals USA </td><td> 209-12.041 </td><td></td></tr><tr><td></td><td></td><td></td><td> Udstyr </td></tr><tr><td> Spand til æglægning </td><td> Rubbermaid kommercielle produkter </td><td> 6308 </td><td></td></tr><tr><td> CL2 IEC centrifuge med svingende spand rotor </td><td> Thermo Scientific </td><td> 004260F </td><td></td></tr><tr><td> HB6 svingende spand rotor </td><td> Thermo Scientific </td><td> 11.860 </td><td></td></tr><tr><td> Sorvall RC6 plus SuperSpeed ​​centrifuge </td><td> Thermo Scientific </td><td> 46.910 </td><td></td></tr><tr><td> UV tværbinder </td><td> UVP </td><td> 95-0174-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> Løsninger </td></tr><tr><td> 1x MMR </td><td></td><td></td><td> 100 mM NaCI, 2 mM KCI, 0,5 mM MgSO4, 2,5 mM CaCl2, 5 mM HEPES, pH indstilles til 7,8 med 10 M NaOH </td></tr><tr><td> Aprotinin / Leupeptin lager </td><td></td><td></td><td> 10 mg / ml hver i vand. Gemme 20 pi alikvoter ved -80 ° C. </td></tr><tr><td> Puffer X </td><td></td><td></td><td> 0,2 M saccharose, 80 mM KCI, 15 mM NaCI, 5 mM MgCl2, 1 mM EDTA, 10 mM HEPES, pH indstilles til 7,5 ved HCI </td></tr><tr><td> Cycloheximid lager </td><td></td><td></td><td> 10 mg / ml i vand. Store 1 ml prøver ved -20 ° C. </td></tr><tr><td> Cytochalasin B bestand </td><td></td><td></td><td> 5 mg / ml i DMSO. Gemme 20 pi alikvoter ved -20 ° C. </td></tr><tr><td> Dithiothreitol (DTT) bestand </td><td></td><td></td><td> 1 M i vand. Store 1 ml prøver ved -20 ° C. </td></tr><tr><td> ELB </td><td></td><td></td><td> 0,25 M saccharose, 1 mM DTT, 50 ug / ml cycloheximid, 2,5 mM MgCl2, 50 mM KCI, 10 mM HEPES, pH7.7 </td></tr><tr><td> Nocodazol lager </td><td></td><td></td><td> 10 mg / ml i DMSO. Store 5 pi alikvoter ved -80 ° C. </td></tr><tr><td> Energi Blanding </td><td></td><td></td><td> 375 mM creatinphosphat, 50 mM ATP og 25 mM MgCl2. Portioner gemmes ved -80 ° C. </td></tr><tr><td> Nuclear farvestofopløsning </td><td></td><td></td><td> 0,4 ug / ml Hoechst 33258, 25% glycerol(Vol / vol), i 1 x PBS </td></tr><tr><td> Tautomycin lager </td><td></td><td></td><td> 100 uM i DMSO. Gemme 10 pi alikvoter ved -80 ° C. </td></tr></tbody></table>

study of the dna damage checkpoint using xenopus egg extracts

På daglig basis, er celler underkastes en række forskellige endogene og miljømæssige fornærmelser. For at bekæmpe disse fornærmelser er celler udviklet DNA beskadigelse checkpoint signalering som en overvågningsmekanisme til at fornemme DNA-skader og direkte cellulære reaktioner på DNA-skader. Der er flere grupper af proteiner kaldet sensorer, transducere og effektorer er involveret i DNA beskadigelse checkpoint&#39;et signalering (fig. 1). I denne komplekse signalvejen, er ATR (ATM og Rad3-relateret) en af ​​de store kinaser der kan reagere på DNA-beskadigelse og replikation stress. Aktiveret ATR kan phosphorylere dens downstream substrater såsom Chk1 (Checkpoint kinase 1). Følgelig phosphoryleret og aktiveret Chk1 fører til mange downstream effekter i DNA beskadigelse checkpoint&#39;et herunder cellecyklusstandsning, transkriptionsaktivering, DNA-beskadigelse reparation, og apoptose eller fremadskridende alderdom (figur 1). Når DNA er beskadiget, ikke at aktivere DNA beskadigelse checkpoint&#39;et resulterer i unrepluftet skade og efterfølgende genomisk ustabilitet. Undersøgelsen af ​​DNA skade checkpoint vil belyse, hvordan cellerne opretholder genomisk integritet og give en bedre forståelse af, hvordan humane sygdomme, såsom kræft, udvikle sig. Xenopus laevis æg ekstrakter dukker op som en kraftfuld cellefri ekstrakt modelsystem i DNA beskadigelse checkpoint&#39;et forskning. Lav hastighed ekstrakt (LSE) blev oprindeligt beskrevet af Masui gruppe 1. Tilsætningen af ​​demembranated sperm chromatin til LSE resulterer i kerner dannelse hvor DNA replikeres i en semi måde gang pr cellecyklus. Den ATR/Chk1-mediated checkpoint signalvejen udløses af DNA-beskadigelse eller replikation stress 2. To metoder anvendes for tiden til at inducere DNA beskadigelse checkpoint: DNA-ødelæggende metoder og DNA-skader der efterligner strukturer 3. DNA-beskadigelse kan induceres af ultraviolet (UV) bestråling, γ-bestråling, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinolin-1-oxid (4-NQO), eller aphidicolin 3, 4. MMS er et alkyleringsmiddel, der inhiberer DNA-replikation og aktiverer ATR/Chk1-mediated DNA beskadigelse checkpoint&#39;et 4-7. UV-bestråling også udløser ATR/Chk1-dependent DNA-skader checkpoint 8. Den DNA-beskadigelse-efterlignende struktur AT70 er et udglødet kompleks af to oligonucleotider poly-(dA) 70 og poly-(dT) 70. Det AT70-systemet blev udviklet i Bill Dunphy laboratorium og er almindeligt anvendt til at fremkalde ATR/Chk1 checkpoint signalering 9-12. Her beskriver vi protokoller (1) til fremstilling af cellefrie ekstrakter æg (LSE), (2) at behandle Xenopus spermakromatin med to forskellige DNA-ødelæggende fremgangsmåder (MMS og UV), (3) til fremstilling af DNA-beskadigelse-efterlignende struktur AT70, og (4) at udløse ATR/Chk1-mediated DNA beskadigelse checkpoint&#39;et i LSE med beskadiget spermakromatin eller en DNA-beskadigelse-efterlignende struktur.

Watch this Scientific Journal Video about Study of the DNA Damage Checkpoint using Xenopus Egg Extracts at JoVE.com

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus æggeekstrakt er et nyttigt model-system til at undersøge DNA beskadigelse checkpoint&#39;et. Denne protokol er til fremstilling af Xenopus æg ekstrakter og DNA beskadigelse checkpoint&#39;et inducerende reagenser. Disse teknikker kan tilpasses til en række DNA-ødelæggende fremgangsmåder i undersøgelsen af ​​DNA beskadigelse checkpoint&#39;et signalering.

Undersøgelse af DNA beskadigelse checkpoint&#39;et ved hjælp<em&gt; Xenopus</em&gt; Æg Ekstrakter

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

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Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

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