Kombineret Immunofluorescens og DNA FISH på 3D-bevaret Interfasen Kerner til Uddannelse Ændringer i 3D Nuclear Organization
Summary
Her beskriver vi en protokol til samtidig påvisning af histon ændringer ved immunofluorescens og DNA sekvenser ved DNA FISK efterfulgt af 3D mikroskopi og analyser (3D immuno-DNA FISH).
Abstract
Fluorescerende in situ hybridisering med DNA-prober på 3-dimensionelt bevarede kerner efterfulgt af 3D konfokal mikroskopi (3D DNA FISH) repræsenterer den mest direkte måde at visualisere placeringen af gen loci, kromosomale subregioner eller hele områder i de enkelte celler. Denne type analyse giver indblik i den globale arkitektur af kernen samt opførsel af specifikke genomiske loci og regioner i det nukleare område. Immunofluorescens på den anden side tillader påvisning af kerneproteiner (modificerede histoner, histon varianter og modifikatorer, transkriptionsmekanisme og faktorer, nukleare delrum osv.). Den største udfordring i at kombinere immunofluorescens og 3D DNA FISH er, på den ene side at bevare epitopen detekteres af antistoffet såvel som 3D arkitektur af kernen, og på den anden side, for at tillade gennemtrængning af DNA-probe til påvisning gen loci eller kromosom territorier 1-5. Her giver vi en protokol, der kombinerer visualisering af kromatin modifikationer med genomiske loci i 3D konserveret kerner.
Introduction
Epigenetiske mekanismer udløser etablering og arv af udviklings-og celletype specifikke transkriptionelle profiler. På et niveau dette indebærer modulering af kromatin emballage, der definerer aktive eller stille genomiske regioner. I en større målestok, også global 3D organisering af genomet og nuklear struktur spiller en rolle i kontrollen af transkriptionelle mønstre. Således dissektion af disse epigenomic funktioner er afgørende for en fuld forståelse af, hvordan generne reguleres 6-11.
Kombineret immunofluorescens og 3D DNA FISH giver en unik mulighed for at supplere molekylære og biokemiske analyser ved at vurdere specifikke interaktioner / sammenslutninger af DNA-sekvenser og / eller proteiner i kernen. Og selv om genom-dækkende high throughput-teknikker, såsom chromatin immunoprecipitation (chip-seq) eller kromosom capture konformation kombineret med dyb sekventering (4C-seq, 5C, Hi-C) giver global daten på cellepopulationer 12, immunofluorescens / DNA FISH teknikker muliggøre analyser på enkelt celle niveau.
Her beskriver vi en protokol til samtidig påvisning af histon ændringer ved immunofluorescens og DNA sekvenser ved DNA FISK efterfulgt af 3D mikroskopi og analyser (3D immuno-FISH). Fordelen ved denne protokol er den kombinerede visualisering af DNA og bevarelse af proteinstrukturer. Vores erfaring på dette område har gjort det muligt for os at forbedre og forenkle eksisterende protokoller. Selv om vi har anvendt denne protokol til påvisning af DNA dobbeltstrengede brud i lymfocytter undergår rekombination, kan denne metode anvendes på andre proteiner og andre celletyper.
Protocol
Representative Results
Discussion
Teknikkerne beskrevet ovenfor blev anvendt i vores laboratorium til analyse reguleringen af V (D) J rekombination af immunoglobulinet og Tcra / d loci i udviklingen af lymfocytter 30,31. Vi tror på, at denne teknik kan tilpasses til påvisning af forskellige nukleare proteiner, nukleare rum og loci, i forskellige celletyper. Ændringer af protokollen kan være nødvendig, og i dette tilfælde de vigtigste trin til at fokusere på, er følgende. For det første kan længden af permeabi…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi vil gerne takke medlemmerne af Skok lab, især Susannah Hewitt, for diskussioner og kommentarer. Dette arbejde er støttet af National Institute of Health tilskud R01GM086852, RC1CA145746 (JAS). JAS er en Leukemia & Lymphoma Society lærd. JC er en Irvington Institute Fellow af Cancer Research Institute. MM er støttet af en National Science Foundation Grant Integrativ Graduate Uddannelse og Forskning praktik (NSF IGERT 0.333.389).
Materials
| Name of Reagent/Material | Company | Catalogue Number | Comments |
| H2O | Fisher | # BP2470 | |
| RNase A | Sigma | # R4642 | |
| dNTP | Sigma | # DNTP100 | |
| Alexa dUTP | Invitrogen | # C11397 to C-11401 | |
| Cy3 or Cy5 dUTP | Fisher | # 45-001-xxx | |
| DNase I | Roche | # 04536282001 | |
| DNA Pol I | Biolabs | # M0209 | |
| 0.025 μm filters | Millipore | # VSWP02500 | |
| Cot-1 DNA 1 mg/ml | Invitrogen | # 18440 | |
| Hybloc DNA 1 mg/ml | Applied Genetics | # MHB | |
| Salmon sperm | Sigma | # D1626 | powder to be resuspended at 10 mg/ml in H2O |
| NaAc (Sodium Acetate, pH 5.2, buffer solution) | Sigma | # S7899 | |
| Ficoll 400 (Mol Biol grade) | Fisher | # 525 | |
| Polyvinylpyrrolidone (Mol Biol grade) | Fisher | # BP431 | |
| Dextran sulfate powder | Sigma | # D8906 | |
| SSPE (Saline-Sodium Phosphate-EDTA) 20x solution | Fisher | # BP1328 | |
| Formamide | Fisher | # BP227 | |
| Coverslips | Fisher | # 12-548-B | |
| Slides | Fisher | # 12-550 | |
| 6-well plates | Fisher | # 0720080 | |
| PBS, 10x | Fisher | # MT-46-013-CM | |
| Poly-L-lysine solution | Sigma | # P8920 | |
| Paraformaldehyde, prills, 95% | Sigma | # 441244 | |
| Triton-X-100, Mol Biol grade | Sigma | # T8787 | |
| BSA (Bovine Serum Albumin) Fraction V | Fisher | # BP 1600 | |
| Normal goat serum | Vector Labs | # S-1000 | |
| Tween-20, Mol Biol grade | Sigma | # P9416 | |
| SSC (Saline Sodium Citrate) 20x solution | Fisher | # BP1325 | |
| ProLong Gold antifade reagent | Invitrogen | # P36930 | |
| DAPI (4′,6-diamidino-2-phenylindole) | Sigma | # D9542 | |
| Best test one coat rubber cement | Art or office supply stores | ||
| Table 1. Specific reagents and small equipment. |
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