Påvisning af 5-Hydroxymethylcytosin i neurale stamceller og hjerner af mus
Summary
Her præsenterer vi en protokol til påvisning af 5-hydroxymethylcytosin i celler og hjernevæv, ved hjælp af immunofluorescens farvning og DNA dot-blot metoder.
Abstract
Der er identificeret flere DNA-modifikationer i pattedyrs genomet. Af det, 5-methylcytosin og 5-hydroxymethylcytosin-medierede epigenetiske mekanismer er blevet intensivt undersøgt. 5-hydroxymethylcytosin viser dynamiske funktioner under embryonale og postnatale udvikling af hjernen, spiller en regulerende funktion i genekspression, og er involveret i flere neurologiske lidelser. Her beskriver vi de detaljerede metoder, herunder immunofluorescens farvning og DNA dot-blot at detektere 5-hydroxymethylcytosin i dyrkede celler og hjernens væv af mus.
Introduction
Epigenetiske modifikationer, herunder DNA modifikation, Histon modifikation og RNA modifikation, har vist sig at spille væsentlige funktioner i forskellige biologiske processer og sygdomme1,2,3, 4 , 5 , 6 , 7. i lang tid, DNA-methylering (dvs., 5-methylcytosin (5-mC)) er blevet betragtet som en meget stabil epigenetisk markør og kan ikke ændres yderligere i genomet. For nylig, det har været konstateret, at 5-mC kunne oxideres til 5-hydroxymethylcytosin (5-HMC) af tet (ti-elleve translocations) familie proteiner, herunder TET1, TET2,og TET3 8,9. Yderligere undersøgelser viser, at 5-HMC kunne tjene som en stabil markør og spille biologiske roller ved at regulere genekspression4,10,11,12.
De nuværende beviser indikerer, at 5-HMC er stærkt beriget i neuronal væv/celler i forhold til andre typer af væv i pattedyr, og udviser dynamiske funktioner under neuronal udvikling13,14. I neuronal system, 5-hmC medierede epigenetiske modifikationer spiller en vigtig rolle i reguleringen af neurale stamceller, neuronal aktivitet, indlæring og hukommelse, og er involveret i flere neurologiske lidelser, herunder RETT syndrom, autisme, Alzheimers sygdom, Huntingtons sygdom, etc.2,13,15,16,17,18,19,20.
Der er flere tilgange til påvisning af 5-HMC i celler og væv14,21,22,23,24. Her beskriver vi to metoder til at påvise eksistensen af 5-hmC og kvantificere det globale niveau af 5-hmC: immunofluorescens farvning og DNA dot-blot. Disse to metoder er bekvemme og følsomme, og er blevet anvendt med succes i tidligere undersøgelser25,26,27,28,29,30. De vigtigste trin i disse to metoder er DNA-denaturering. For immunofluorescens farvning af 5-hmC kræves forbehandling af prøver med 1 M HCl. For 5-hmC dot-blot er DNA-denaturering udført med NaOH-opløsning. Disse to metoder sammen med næste generations sekvensering er meget nyttige værktøjer til at undersøge funktionen af 5-hmC.
Protocol
Representative Results
Discussion
Epigenetiske modifikationer spiller vigtige roller under hjernens udvikling, modning og funktion. Som en stabil markør for DNA modifikation reagerer Dynamic 5-hmC på adfærds tilpasning, neuronal aktivitet og er positivt korreleret med genekspression; således er det involveret i den normale funktion af hjernen og neurologiske lidelse4. For at undersøge dets funktion i celler og væv, er det nødvendigt at påvise eksistensen af 5-hmC og sammenligne niveauet før og efter behandling. Her demons…
Disclosures
The authors have nothing to disclose.
Acknowledgements
XL blev delvist støttet af det nationale Key R & D program i Kina (2017YFE0196600) og National Natural Science Foundation of China (Grant NOS. 31771395, 31571518). Q.S. blev støttet af Kinas nationale nøgle forsknings-og udviklingsprogram (2017YFC1001703) og det centrale forsknings-og udviklingsprogram for Zhejiang-provinsen (2017C03009). W.X. blev støttet af den naturvidenskabelige Foundation i Zhejiang-provinsen (LY18H020002) og Science Technology Department of Zhejiang-provinsen (2017C37057).
Materials
| 4'-6-diamidino-2-phenylindole (DAPI ) | Sigma-Aldrich | D8417 |
| Adobe Photoshop software | Adobe Inc. | / |
| Alexa Fluor 488 goat anti-rabbit IgG | Thermo Fisher | A11008 |
| Alexa Fluor 568 goat anti-mouse IgG | Thermo Fisher | A11001 |
| anti-5-hydroxymethylcytosine | Active Motif | 39769 |
| anti-NeuN | Millipore | MAB377 |
| B27 supplement | Gibco | 12587-010 |
| B27 supplement | Gibco | 12580-010 |
| B27 supplement | Gibco | 17504-044 |
| Cryostat microtome | Leica | CM1950 |
| DMEM/F-12 medium | OmegaScientific | DM25 |
| epidermal growth factor | PeproTech | 100-15 |
| Fibroblast growth factor-basic | PeproTech | 100-18B |
| forskolin | Sigma-Aldrich | F6886 |
| GlutaMax | Thermo | 35050061 |
| L-Glutamine | Gibco | 25030-149 |
| neurobasal medium | Gibco | 21103-049 |
| normal goat serum | Vector Laboratories | Z0325 |
| nylon membrane (Hybond™-N+ ) | Amersham Biosciences | RPN303B |
| OCT | Leica | 14020108926 |
| Pen Strep | Gibco | 15140-122 |
| phenol: chloroform: isoamyl alcohol (25: 24:1 ) | Sigma-Aldrich | 516726 |
| Poly-D-Lysine | Sigma | P0899-10 |
| proteinase K | VVR | 39450-01-6 |
| retinoic acid | Sigma-Aldrich | R2625 |
| Triton X-100 | Solarbio | T8210 |
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