Name: chromosome replicating timing combined with fluorescent in situ hybridization
Uploaded: 2012-12-10T13:00:00
Description: Watch this Scientific Journal Video about Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization at JoVE.com

Dette arbejde blev støttet af en bevilling fra National Cancer Institute, CA131967.

1. BrdU Indbygning (Terminal Mærkning) <ol><li> Plade celler til omkring 70% konfluens i en 150 mm vævskulturskål 24 timer inden tilsætningen af ​​BrdU. </li><li> Erstatte medierne med frisk komplet medium indeholdende 20 ug / ml BrdU (Sigma) ved passende tidspunkter før høst. Hvor længe celler dyrkes i medier med BrdU vil variere med celletypen og arter, typisk G2 fase varer mellem 2 og 5 timer (figur 1). </li></ol> 2. Kromosom Høst af enkeltlagskulturer cellekulture...

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	<li>Payer, B., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X+chromosome+dosage+compensation:+how+mammals+keep+the+balance.">X chromosome dosage compensation: how mammals keep the balance.</a> Annu. Rev. Genet. 42, 733-772 (2008).</li><li>Breger, K. S., Smith, L., Turker, M. S., Thayer, M. 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Fremstillingen af ​​kromosom spreads er et kritisk skridt for en vellykket replikation-timing assayet beskrevet her. Inddragelsen af ​​et colcemid forbehandling trin forud for hypotonisk behandling kan hjælpe i frekvens og spredning af mitotiske celler. Vi typisk udsætte celler for colcemdi for 1-3 timer før høst, og anvendelse Colcemid i en slutkoncentration på 10 ug / ml. Imidlertid kan medtagelse af et colcemid forbehandlingstrin ændre længden af G2 og derfor kan ændre den åbenbare replikation timing...

<table border="1"><tbody><tr><td> Navn reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> Anti-BrdU-FITC </td><td> Roche Millipore </td><td> 11202593001 MAB326F </td><td> 50 pg / pl </td></tr><tr><td> Nick translation kit </td><td> Abbott Molekylær (Vysis) </td><td> 07J00-0001 </td><td></td></tr><tr><td> Spectrum Orange dUTP </td><td> Abbott Molekylær (Vysis) </td><td> 02N33-050 </td><td></td></tr><tr><td> CEP </td><td> Abbott Molekylær (Vysis) </td><td> Varierer </td><td></td></tr><tr><td> LSI / WCP hybridiseringsbuffer </td><td> Abbott Molekylær (Vysis) </td><td> 06J67-011 </td><td></td></tr><tr><td> CEP hybridiseringsbuffer </td><td> Abbott Molekylær (Vysis) </td><td> 07J36-001 </td><td></td></tr><tr><td> Kromosom paints </td><td> MetaSystems Group </td><td> D-14NN-050-TR </td><td></td></tr><tr><td> Olympus BX61 fluorescerende mikroskop </td><td> Olympus </td><td> BX61TRF-1-5 </td><td></td></tr><tr><td> Mikroskop imaging software system </td><td> Applied Imaging </td><td> Cytovision 3.93.1 </td><td></td></tr><tr><td> Digital Camera </td><td> Olympus </td><td> UCMAD3 </td><td></td></tr><tr><td></td><td></td><td></td><td> In situ hybridisering OPSKRIFTER Formamid Solutions 70% Formamide/2x SSC 35 ml formamid * (Sigma) 10 ml 10x SSC 5 ml d 2 H 2 0 pH til 7,0 med HCI (Sigma) * Det er vigtigt at bruge formamid som er blevet opbevaret ved -20 ° C. Langvarig opbevaring ved stuetemperatur vil generere myresyre, og pH wdårligt være for lav. 50% Formamide/2x SSC 25 ml formamid (Sigma) 10 ml 10x SSC 15 ml dH2O pH til 7,0 med HCI (Sigma) 20x SSC, 4 L 702 g NaCI (Sigma) 358 g Na-citrat (Sigma) dH 2 O til volumen PN Buffer [0,1 M NaP0 4 0.1% NP_40 (Sigma)] Foretag en 0,1 M opløsning hver af natriumphosphat (Filter steriliseres og opbevares i 500 ml alikvoter). 0,1 M NaH 2 P0 4, 1 L 13,8 g NaH 2 P0 4 (Sigma): dH 2 O til volumen 0,1 M NaH 2 P0 4 1 L </strong&gt; 14,2 g NaH 2 P0 4 (Sigma) dH 2 O til volumen. PN: Indstil pH-værdi på 0,1 M Na 2 HP0 4 til pH 8,0 med 0,1 M NaH 2 P0 4. Filter sterilisere, og der tilsættes 1 ml af NP-40. PNM 50 ml 1,25 g ikke-fedtholdig tørmælk (Sigma) 25 ml PN-puffer (opskrift ovenfor) Bland i 15-20 minutter under konstant omrøring. Spin 2 gange ved 400 x g i 10 minutter. Brug supernatanten, og sørg for ikke at forstyrre udfældede mælkeproteiner. </td></tr></tbody></table>

chromosome replicating timing combined with fluorescent in situ hybridization

Mammalian DNA-replikation initierer flere steder langs kromosomer på forskellige tidspunkter i løbet af S-fasen, efter en tidsmæssig replikation program. Specifikationen af ​​replikation timing menes at være en dynamisk proces, reguleret af vævsspecifikke og udviklingsmæssige signaler, som reagerer på epigenetiske modifikationer. Men de mekanismer, der regulerer, hvor og hvornår DNA-replikation starter langs kromosomerne stadig dårligt forstået. Homologe kromosomer normalt replikere synkront, men der er markante undtagelser fra denne regel. For eksempel en af de to X-kromosomer hos kvindelige pattedyrceller bliver forsinket replikerende gennem en proces, der kaldes X-inaktivering 1. Sammen med denne forsinkelse i replikation timing, skønnes at være 2-3 timer, bliver størstedelen af ​​gener transkriptionelt tavs på én X-kromosom. Desuden regulerer en diskret cis-virkende locus, kendt som X-inaktivering er dette X inaktiveringsproces, herunder the induktion af forsinket replikation timing på hele inaktive X-kromosom. Desuden udsættes visse kromosomomlejringer fundet i cancerceller og i celler for ioniserende stråling vise en væsentlig forsinkelse i replikation timingen af&gt; 3 timer, der påvirker hele kromosom 2,3. Seneste arbejde fra vores laboratorium viser, at afbrydelse af diskrete cis-agerende autosomal loci resulterer i et meget sent replikerende fænotype, der påvirker hele kromosom 4. Yderligere &#39;kromosom engineering &quot;undersøgelser viser, at visse kromosomomlejringer påvirker mange forskellige kromosomer resultat i denne unormale replikations-timing fænotype, hvilket tyder på, at alle pattedyrs-kromosomer indeholder diskrete cis-virkende loci, der styrer korrekt replikation timingen af individuelle chromosomer 5. Her præsenteres en metode til kvantitativ analyse af kromosomreplikation timing kombineret med fluorescerende in situ hybridisering. DetteFremgangsmåden muliggør en direkte sammenligning af replikation timing mellem homologe kromosomer i samme celle, og blev tilpasset fra 6. Derudover muliggør denne fremgangsmåde til utvetydig identifikation af kromosomale omlejringer, der korrelerer med ændringer i replikation timing, der påvirker hele kromosomet. Denne metode har fordele i forhold til nyligt udviklede high throughput mikro-array eller sekventering protokoller, der ikke kan skelne mellem homologe alleler stede på omarrangeret og un-omarrangeret kromosomer. Endvidere, fordi den her beskrevne fremgangsmåde evalueres enkelte celler kan detektere ændringer i kromosomreplikation timing for kromosomale omlejringer, der er til stede i kun en fraktion af cellerne i en population.

Et eksempel på replikationen timing analyse for humant kromosom 6 er vist i figur 2. Celler, der indeholder en deletion af ASAR6 gen 4, placeret på 6q16.1, blev udsat for BrdU i 5 timer, høstet til mitotiske celler og behandlet til FISH med en kromosom 6 maling probe (Vysis) og for BrdU-inkorporering. Bemærk, at der er en signifikant forskel i BrdU båndmønster mellem de to 6 s, hvilket er konsistent med en forsinkelse i replikation hvornår&gt; 2 timer for en af kromosom 6 er [se 4...

Watch this Scientific Journal Video about Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization at JoVE.com

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

En kvantitativ metode til analyse af kromosomreplikation timing er beskrevet. Fremgangsmåden anvender BrdU-inkorporering i kombination med fluorescerende<em&gt; In situ</em&gt; Hybridisering (FISH) til at vurdere replikation timingen af ​​mammale kromosomer. Denne teknik giver mulighed for direkte sammenligning af omlejrede og un-omlejrede kromosomer i samme celle.

Kromosom Replikerer Timing Kombineret med Fluorescent<em&gt; In situ</em&gt; Hybridisering

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

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Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

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