Name: Engenharia Epigenética de Células K562: Estratégia Episómica de Vetor Duplo para Metilação de DNA Direcionada Estável usando Proteínas de Fusão dCas9-DNMT3A e -HDAC1 (Vídeo) | JoVE Journal
Uploaded: 2025-10-31T14:30:00.000+00:00
Duration: 9 min 56 s
Description: Aprenda a realizar edição epigenética direcionada em células K562 usando um sistema CRISPR/dCas9 episômico com proteínas de fusão dCas9-DNMT3A e dCas9-HDAC1. Este método permite metilação de DNA estável e específica de locus com efeitos off-target reduzidos.

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5 minutes

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</ol>

<table><tbody><tr><td>Mistura Mestre Digital PCR Absolute Q DNA (5x)</td><td>Biossistemas Aplicados</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>Separador de Células Melodias BD FACS</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>Mistura de Solução de Desoxinucleotídeos (dNTP) (10 mM cada)</td><td>NEB</td><td>N0447</td><td></td></tr><tr><td>DMEM Glicose Alta com Glutamina estável, com meio de Piruvato de Sódio</td><td>Biowest</td><td>L0103</td><td></td></tr><tr><td>DNA polimerase I, Fragmento Grande (Klenow)</td><td>NEB</td><td>M0210</td><td></td></tr><tr><td>DNMT3A (CD), sequências HDAC1</td><td>IDT</td><td></td><td></td></tr><tr><td>DreamTaq DNA Polimerase&nbsp;</td><td>Termo Científica</td><td>EP0712</td><td></td></tr><tr><td>EpiTect</td><td>Qiagen</td><td>59104</td><td>Kit de conversão de bissulfito &nbsp;</td></tr><tr><td>Kit de bisulfito Epitect</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>Soro Fetal Bovino</td><td>Gibco</td><td>A5256701</td><td></td></tr><tr><td>Antibiótico seletivo de geneticina (G418 sulfato)</td><td>Gibco</td><td>11811031</td><td></td></tr><tr><td>Kit de transfecção Lipofectamine 3000</td><td>Invitrogen</td><td>L3000015</td><td></td></tr><tr><td>NEB&reg; &nbsp; 5-alfa Competent&nbsp;E. coli&nbsp; (Alta eficiência)</td><td>NEB</td><td>C2987I</td><td></td></tr><tr><td>NEB&reg; &nbsp; 5-alfa F'Iq&nbsp; Competent&nbsp;E. coli&nbsp; (Alta eficiência)</td><td>NEB</td><td>C2992H</td><td></td></tr><tr><td>Tecido Nucleospin &nbsp;</td><td>Macherey-Nagel</td><td>740952.50</td><td></td></tr><tr><td>PBS, solução 10x pH 7,4</td><td>Lonza</td><td>BE17-517Q</td><td></td></tr><tr><td>Solução de penicilina-estreptomicina 100x</td><td>Biowest</td><td>L0022</td><td></td></tr><tr><td>Vetor pGuide</td><td>OriGene Technologies</td><td>GE100042</td><td></td></tr><tr><td>Fenol:Clorofórmio:Isoamílico Álcool 25:24:1 Saturado com 10 mM Tris, pH 8,0, 1 mM de EDTA</td><td>Sigma-Aldrich</td><td>P2069</td><td></td></tr><tr><td>Primers, sgRNAs e nbsp;</td><td>Eurofinas</td><td></td><td></td></tr><tr><td>Projeto de Ensaio PyroMark &nbsp;</td><td>Qiagen</td><td>Versão 2.0</td><td>Ferramenta de design de primer de bisulfito &nbsp;</td></tr><tr><td>Reagentes PyroMark Gold Q24</td><td>Qiagen</td><td>970802</td><td></td></tr><tr><td>Kit de PCR PyroMark & nbsp;</td><td>Qiagen</td><td>978703</td><td></td></tr><tr><td>PyroMark Q24 MDx</td><td>Qiagen</td><td></td><td>Estação de trabalho de pirosequenciamento&nbsp;</td></tr><tr><td>PyroMark Q24 Software&nbsp;</td><td>Qiagen</td><td>v2.0.8</td><td>Software de pirosequenciamento &nbsp;</td></tr><tr><td>Q5&reg; DNA polimerase de alta fidelidade</td><td>NEB</td><td>M0491</td><td></td></tr><tr><td>Kit de Transcrição Reversa QuantiTect</td><td>Qiagen</td><td>205311</td><td></td></tr><tr><td>Kit de Placa QuantStudio Absolute Q MAP16</td><td>Biossistemas Aplicados</td><td>A52865</td><td>Sistema de PCR Digital &nbsp;</td></tr><tr><td>SYBR Green I Nucleic Acid Gel Stain, concentrado 10.000x em 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>Polinucleotídeo quinase T4</td><td>NEB</td><td>M0201</td><td></td></tr><tr><td>Reagente Trizol</td><td>Invitrogen</td><td>15596026</td><td></td></tr></tbody></table>

engenharia epigenética de células k562: estratégia episómica de vetor duplo para metilação de dna direcionada estável usando proteínas de fusão dcas9-dnmt3a e -hdac1

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

Engenharia Epigenética de Células K562: Estratégia Episómica de Vetor Duplo para Metilação de DNA Direcionada Estável usando Proteínas de Fusão dCas9-DNMT3A e -HDAC1

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

Bioengenharia

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Eficiente Geração células iPS a partir de sangue, utilizando epissomas e HDAC Inibidores

Descrevemos em detalhes dois protocolos de base química para cultivo de células-tronco embrionárias do mouse. Esse novo método utiliza mecanismos sinérgicos de promover oxidação mediada por Tet (vitamina C) e reprimindo a síntese de novo de 5-metilcitosina (por PD0325901) para manter o DNA hypomethylation e pluripotência das células-tronco embrionárias do mouse.

Um método alternativo de cultura para manter Hypomethylation genômico das células-tronco embrionárias Mouse usando MEK inibidor PD0325901 e vitamina C

Protocolos detalhados para a introdução de uma enzima de divisão de engenharia-TET2 (cidra) em células de mamíferos para química inducible DNA hydroxymethylation e remodelação epigenéticas são apresentados.

Uma enzima de Split-TET2 projetado para Hydroxymethylation de DNA químico-inducible e remodelação epigenéticas

Regulamento do ambiente da cromatina é um processo essencial, necessário para a expressão do gene apropriado. Aqui, descrevemos um método para controlar a expressão do gene através do recrutamento de cromatina-modificando máquinas de forma gene-específico e reversível.

Reprimir a transcrição do Gene redirecionando maquinaria celular com modificadores epigenéticas químicas

Alvo de DNA Epigenoma edição representa uma poderosa abordagem terapêutica. Este protocolo descreve a produção, purificação e concentração de vetores Lentivirus all-in-one, abrigando o transgene CRISPR-dCas9-DNMT3A para aplicativos de edição de Epigenoma em células-tronco pluripotentes induzidas humana (hiPSC)-derivado de neurônios.

Vetor de Lentivirus de plataforma para a entrega eficiente de ferramentas de edição de Epigenoma em humanos induzida por modelos de doenças derivadas de células-tronco pluripotentes

Apresentamos aqui um método que combina o uso de apagamento epigenético químico com pistas relacionadas à mecanosensagem para gerar eficientemente células pluripotentes de mamíferos, sem a necessidade de transfecção genética ou vetores retrovirais. Essa estratégia é, portanto, promissora para a medicina translacional e representa um notável avanço na tecnologia organoide de células-tronco.

Uma estratégia de duas etapas que combina modificação epigenética e sinais biomecânicos para gerar células pluripotentes mamíferas

Aqui, apresentamos um protocolo para a seleção in vitro de repressores transcricionais projetados (ETRs) com alta, longa duração, estável, eficiência de silenciamento no alvo e baixa atividade fora do alvo em todo o genoma. Esse fluxo de trabalho permite reduzir um repertório inicial e complexo de ETRs candidatos a uma lista curta, adequada para avaliação adicional em cenários terapeuticamente relevantes.

In vitro Seleção de Repressores Transcricionais Projetados para Silenciamento Epigenético Direcionado

Novas estratégias para modelar fielmente mutações somáticas em células-tronco hematopoiéticas e progenitoras (HSPCs) são necessárias para melhor estudar a biologia das células-tronco hematopoiéticas e as neoplasias hematológicas. Aqui, um protocolo para modelar mutações heterozigotas de ganho de função em HSPCs combinando o uso de CRISPR/Cas9 e transdução dupla de doador de rAAV é descrito.

Engenharia de Mutações de Ganho de Função Heterozigotas Oncogênicas em Células-Tronco Hematopoiéticas Humanas e Progenitoras

O protocolo descreve métodos para edição de epigenoma baseada em Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) em linhagens de células humanas usando transfecção de DNA de plasmídeo e nucleofecção de mRNA.

Edição de Epigenoma CRISPR em Células Humanas usando Transfecção de DNA de Plasmídeo e Entrega de Nucleofecção de mRNA

Um protocolo detalhado é fornecido para o uso da tecnologia CRISPR / Cas9 para obter knock-in direcionado altamente eficiente de grandes construções multicistrônicas em células T humanas primárias por meio da via de reparo de DNA de junção de extremidade mediada por homologia (HMEJ). As células T projetadas com este protocolo adaptável ao cGMP mantêm excelente expansão celular, citotoxicidade e produção de citocinas.

Engenharia não viral de células T humanas primárias por meio de integração direcionada de junção de extremidade mediada por homologia de grandes modelos de DNA

Aprenda a realizar edição epigenética direcionada em células K562 usando um sistema CRISPR/dCas9 episômico com proteínas de fusão dCas9-DNMT3A e dCas9-HDAC1. Este método permite metilação de DNA estável e específica de locus com efeitos off-target reduzidos.

Engenharia Epigenética de Células K562: Estratégia Episómica de Vetor Duplo para Metilação de DNA Direcionada Estável usando Proteínas de Fusão dCas9-DNMT3A e -HDAC1 (Vídeo) | JoVE Journal

Engenharia Epigenética de Células K562: Estratégia Episómica de Vetor Duplo para Metilação de DNA Direcionada Estável usando Proteínas de Fusão dCas9-DNMT3A e -HDAC1

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