Name: Epigenetische manipulatie van K562-cellen: dual-vector episomale strategie voor stabiele gerichte DNA-methylering met behulp van dCas9-DNMT3A en -HDAC1 fusie-eiwitten (Video) | JoVE Journal
Uploaded: 2025-10-31T14:30:00.000+00:00
Duration: 9 min 56 s
Description: Leer een stabiele, niet-integrerende methode voor gerichte DNA-methylering in K562-cellen met behulp van een dual-vector CRISPR/dCas9-systeem. Dit protocol combineert dCas9-DNMT3A en dCas9-HDAC1 effectoren voor duurzame epigenetische modificatie met verminderde off-target effecten.

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5 minutes

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<table><tbody><tr><td>Absolute Q DNA Digital PCR Master Mix (5x)</td><td>Applied Biosystems</td><td>A52490</td><td></td></tr><tr><td>AgeI-HF&nbsp;</td><td>NEB</td><td>R3552</td><td></td></tr><tr><td>AscI</td><td>NEB</td><td>R0558</td><td></td></tr><tr><td>AseI</td><td>NEB</td><td>R0526</td><td></td></tr><tr><td>BamHI-HF</td><td>NEB</td><td>R3136</td><td></td></tr><tr><td>BD FACS Melody Cell Sorter</td><td>BD</td><td></td><td></td></tr><tr><td>Bglll</td><td>NEB</td><td>R0144</td><td></td></tr><tr><td>BspEI</td><td>NEB</td><td>R0540</td><td></td></tr><tr><td>Deoxynucleotide (dNTP) Solution Mix (10 mM each)</td><td>NEB</td><td>N0447</td><td></td></tr><tr><td>DMEM High Glucose w/stable Glutamine, w/Sodium Pyruvate medium</td><td>Biowest</td><td>L0103</td><td></td></tr><tr><td>DNA Polymerase I, Large (Klenow) Fragment</td><td>NEB</td><td>M0210</td><td></td></tr><tr><td>DNMT3A (CD), HDAC1 sequences</td><td>IDT</td><td></td><td></td></tr><tr><td>DreamTaq DNA Polymerase&nbsp;</td><td>Thermo Scientific</td><td>EP0712</td><td></td></tr><tr><td>EpiTect</td><td>Qiagen</td><td>59104</td><td>Bisulfite conversion kit&nbsp;</td></tr><tr><td>Epitect Bisulfite kit</td><td>Qiagen</td><td>59104</td><td></td></tr><tr><td>Esp3I</td><td>NEB</td><td>R0734</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>A5256701</td><td></td></tr><tr><td>Geneticin selective antibiotic (G418 sulfate)</td><td>Gibco</td><td>11811031</td><td></td></tr><tr><td>Lipofectamine 3000 transfection kit</td><td>Invitrogen</td><td>L3000015</td><td></td></tr><tr><td>NEB&reg;&nbsp;5-alpha Competent&nbsp;E. coli&nbsp;(High Efficiency)</td><td>NEB</td><td>C2987I</td><td></td></tr><tr><td>NEB&reg;&nbsp;5-alpha F'Iq&nbsp;Competent&nbsp;E. coli&nbsp;(High Efficiency)</td><td>NEB</td><td>C2992H</td><td></td></tr><tr><td>Nucleospin Tissue&nbsp;</td><td>Macherey-Nagel</td><td>740952.50</td><td></td></tr><tr><td>PBS, 10x solution pH 7.4</td><td>Lonza</td><td>BE17-517Q</td><td></td></tr><tr><td>Penicillin-Streptomycin solution 100x</td><td>Biowest</td><td>L0022</td><td></td></tr><tr><td>pGuide vector</td><td>OriGene Technologies</td><td>GE100042</td><td></td></tr><tr><td>Phenol:Chloroform:Isoamyl Alcohol 25:24:1 Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA</td><td>Sigma-Aldrich</td><td>P2069</td><td></td></tr><tr><td>Primers, sgRNAs&nbsp;</td><td>Eurofins</td><td></td><td></td></tr><tr><td>PyroMark Assay Design&nbsp;</td><td>Qiagen</td><td>Version 2.0</td><td>Bisulfite primer design tool&nbsp;</td></tr><tr><td>PyroMark Gold Q24 Reagents</td><td>Qiagen</td><td>970802</td><td></td></tr><tr><td>PyroMark PCR kit&nbsp;</td><td>Qiagen</td><td>978703</td><td></td></tr><tr><td>PyroMark Q24 MDx</td><td>Qiagen</td><td></td><td>Pyrosequencing workstation&nbsp;</td></tr><tr><td>PyroMark Q24 Software&nbsp;</td><td>Qiagen</td><td>v2.0.8</td><td>Pyrosequencing software&nbsp;</td></tr><tr><td>Q5&reg; High-Fidelity DNA Polymerase</td><td>NEB</td><td>M0491</td><td></td></tr><tr><td>QuantiTect Reverse Transcription Kit</td><td>Qiagen</td><td>205311</td><td></td></tr><tr><td>QuantStudio Absolute Q MAP16 Plate Kit</td><td>Applied Biosystems</td><td>A52865</td><td>Digital PCR system&nbsp;</td></tr><tr><td>SYBR Green I Nucleic Acid Gel Stain, 10,000x concentrate in DMSO</td><td>Invitrogen</td><td>S7563</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>NEB</td><td>M0202</td><td></td></tr><tr><td>T4 Polynucleotide Kinase</td><td>NEB</td><td>M0201</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Invitrogen</td><td>15596026</td><td></td></tr></tbody></table>

epigenetische manipulatie van k562-cellen: dual-vector episomale strategie voor stabiele gerichte dna-methylering met behulp van dcas9-dnmt3a en -hdac1 fusie-eiwitten

Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins

Epigenetische manipulatie van K562-cellen: dual-vector episomale strategie voor stabiele gerichte DNA-methylering met behulp van dCas9-DNMT3A en -HDAC1 fusie-eiwitten

Investigating the precise role of DNA methylation in gene transcriptional regulation and developing therapies targeting specific gene methylation patterns ...

epigenetic-engineering-k562-cells-dual-vector-episomal-strategy-for

Research

JoVE Journal

Bioengineering

Bio-engineering

Hier beschrijven we een protocol voor het genereren van humane geïnduceerde pluripotente stamcellen uit perifeer bloed met behulp van een episoom gebaseerd herprogrammering strategie en histondeacetylaseremmers.

Efficiënte iPS Cell Generation van Blood behulp episomen en HDAC-remmers

Wij beschreven twee chemische-gebaseerde protocollen voor het kweken van muis embryonale stamcellen in detail. Deze nieuwe methode maakt gebruik van synergetische mechanismen ter bevordering van Tet-gemedieerde oxidatie (door vitamine C) en onderdrukken van DOVO synthese van 5-methylcytosine (door PD0325901) om DNA hypomethylation en pluripotent van muis embryonale stamcellen te onderhouden.

Een alternatieve cultuur methode om Genomic Hypomethylation van muis embryonale stamcellen met MEK-remmer PD0325901 en vitamine C

Gedetailleerde protocollen voor de invoering van een gemanipuleerde split-TET2 enzym (appelwijn) in zoogdiercellen voor chemische afleidbare DNA-hydroxymethylation en epigenetische remodeling worden gepresenteerd.

Een gemanipuleerde Split-TET2 enzym voor chemische-afleidbare DNA Hydroxymethylation en epigenetische Remodeling

Verordening van de chromatine-milieu is een essentieel proces nodig is voor goede genexpressie. Hier beschrijven we een methode voor het beheersen van de genexpressie door de aanwerving van chromatine-aanpassen machines gen-specifieke en omkeerbare wijze.

Onderdrukken van Gene transcriptie door cellulaire machines met chemische epigenetische Modifiers omleiden

Gerichte DNA epigenome bewerken vertegenwoordigt een krachtige therapeutische aanpak. Dit protocol beschrijft de productie, reiniging en concentratie van de alles-in-één lentivirale vectoren herbergen van de CRISPR-dCas9-DNMT3A-transgenic voor bewerken van epigenome's in menselijke geïnduceerde pluripotente stamcel (hiPSC)-afgeleid van neuronen.

Lentivirale Vector Platform voor de efficiënte levering van Epigenome-bewerkingsgereedschap in mens geïnduceerde pluripotente stamcel afkomstige ziekte modellen

We presenteren hier een methode die het gebruik van chemische epigenetische wissing combineert met mechanosensing-gerelateerde signalen om efficiënt pluripotente cellen van zoogdieren te genereren, zonder de noodzaak van gentransfectie of retrovirale vectoren. Deze strategie is daarom veelbelovend voor translationele geneeskunde en vertegenwoordigt een opmerkelijke vooruitgang in stamcelorganoïde technologie.

Een strategie in twee stappen die epigenetische modificatie en biomechanische signalen combineert om pluripotente cellen van zoogdieren te genereren

Hier presenteren we een protocol voor de in vitro selectie van gemanipuleerde transcriptionele repressoren (ETR's) met een hoge, langdurige, stabiele, on-target uitschakelingsefficiëntie en een lage genoombrede, off-target activiteit. Deze workflow maakt het mogelijk om een initieel, complex repertoire van kandidaat-ETR's terug te brengen tot een shortlist, geschikt voor verdere evaluatie in therapeutisch relevante settings.

In vitro Selectie van gemanipuleerde transcriptionele repressoren voor gerichte epigenetische uitschakeling

Nieuwe strategieën om somatische mutaties in hematopoietische stam- en voorlopercellen (HSPC's) getrouw te modelleren, zijn nodig om de hematopoëtische stamcelbiologie en hematologische maligniteiten beter te bestuderen. Hier wordt een protocol beschreven om heterozygote gain-of-function mutaties in HSPC's te modelleren door het gebruik van CRISPR/Cas9 en dual rAAV donortransductie te combineren.

Engineering oncogene heterozygote gain-of-function mutaties in menselijke hematopoietische stam- en voorlopercellen

Het protocol beschrijft methoden voor geclusterde, regelmatig tussenliggende korte palindromale herhalingen (CRISPR)-gebaseerde epigenoombewerking in menselijke cellijnen met behulp van plasmide-DNA-transfectie en mRNA-nucleofectie.

CRISPR-epigenoombewerking in menselijke cellen met behulp van plasmide-DNA-transfectie en mRNA-nucleofectieafgifte

Er is een gedetailleerd protocol voorzien voor het gebruik van CRISPR/Cas9-technologie om zeer efficiënte, gerichte knock-in van grote, multicistronische constructies in primaire menselijke T-cellen te bereiken via de homologie-gemedieerde eindjoining (HMEJ) DNA-reparatieroute. T-cellen die met dit cGMP-aanpasbare protocol zijn ontwikkeld, behouden een uitstekende celexpansie, cytotoxiciteit en cytokineproductie.

Niet-virale engineering van primaire menselijke T-cellen via homologie-gemedieerde end-joining gerichte integratie van grote DNA-sjablonen

Leer een stabiele, niet-integrerende methode voor gerichte DNA-methylering in K562-cellen met behulp van een dual-vector CRISPR/dCas9-systeem. Dit protocol combineert dCas9-DNMT3A en dCas9-HDAC1 effectoren voor duurzame epigenetische modificatie met verminderde off-target effecten.

Epigenetische manipulatie van K562-cellen: dual-vector episomale strategie voor stabiele gerichte DNA-methylering met behulp van dCas9-DNMT3A en -HDAC1 fusie-eiwitten (Video) | JoVE Journal

Epigenetische manipulatie van K562-cellen: dual-vector episomale strategie voor stabiele gerichte DNA-methylering met behulp van dCas9-DNMT3A en -HDAC1 fusie-eiwitten

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