Name: K562 Hücrelerinin Epigenetik Mühendisliği: dCas9-DNMT3A ve -HDAC1 Füzyon Proteinleri Kullanılarak Stabil Hedefli DNA Metilasyonu için Çift Vektörlü Epizomal Strateji (Video) | JoVE Journal
Uploaded: 2025-10-31T14:30:00.000+00:00
Duration: 9 min 56 s
Description: Keşfedin K562 hücrelerinde dCas9-DNMT3A ve dCas9-HDAC1 füzyon proteinleriyle hedefli DNA metilasyonu için çift vektörlü epizomal CRISPR stratejisi. Bu yöntem stabil epigenetik düzenleme sağlar.

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<table><tbody><tr><td>Absolute Q DNA Dijital PCR Master Mix (5x)</td><td>Uygulamalı Biyosistemler</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>Deoksinükleotid (dNTP) Çözelti Karışımı (her biri 10 mM)</td><td>NEB</td><td>N0447</td><td></td></tr><tr><td>DMEM Yüksek Glikoz, stabil Glutamin ve Sodyum Piruvat ortamı ile</td><td>Biowest</td><td>L0103</td><td></td></tr><tr><td>DNA Polimeraz I, Büyük (Klenow) Parça</td><td>NEB</td><td>M0210</td><td></td></tr><tr><td>DNMT3A (CD), HDAC1 dizileri</td><td>IDT</td><td></td><td></td></tr><tr><td>DreamTaq DNA Polimeraz&nbsp;</td><td>Termo Bilimsel</td><td>EP0712</td><td></td></tr><tr><td>EpiTect</td><td>Qiagen</td><td>59104</td><td>Bisülfit dönüşüm kiti&nbsp;</td></tr><tr><td>Epitect Bisülfit Kiti</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 Sığır Serumu</td><td>Gibco</td><td>A5256701</td><td></td></tr><tr><td>Geneticin seçici antibiyotik (G418 sülfat)</td><td>Gibco</td><td>11811031</td><td></td></tr><tr><td>Lipofektamin 3000 transfeksiyon kiti</td><td>Invitrogen</td><td>L3000015</td><td></td></tr><tr><td>NEB&reg; &nbsp; 5-alfa Yetkin&nbsp;E. coli&nbsp; (Yüksek Verimlilik)</td><td>NEB</td><td>C2987I</td><td></td></tr><tr><td>NEB&reg; &nbsp; 5-alfa F'Iq&nbsp; Yetkin&nbsp;E. coli&nbsp; (Yüksek Verimlilik)</td><td>NEB</td><td>C2992H</td><td></td></tr><tr><td>Nukleospin Dokusu&nbsp;</td><td>Macherey-Nagel</td><td>740952.50</td><td></td></tr><tr><td>PBS, 10x çözelti pH 7.4</td><td>Lonza</td><td>BE17-517Q</td><td></td></tr><tr><td>Penisillin-Streptomisin çözeltisi 100x</td><td>Biowest</td><td>L0022</td><td></td></tr><tr><td>pGuide vektörü</td><td>OriGene Teknolojileri</td><td>GE100042</td><td></td></tr><tr><td>Fenol:Kloroform:İzoamil Alkol 25:24:1 10 mM Tris ile doymuş, pH 8.0, 1 mM EDTA</td><td>Sigma-Aldrich</td><td>P2069</td><td></td></tr><tr><td>Primerler, sgRNA'lar&nbsp;</td><td>Eurofins</td><td></td><td></td></tr><tr><td>PyroMark Assay Tasarımı&nbsp;</td><td>Qiagen</td><td>Sürüm 2.0</td><td>Bisülfit primer tasarım aracı&nbsp;</td></tr><tr><td>PyroMark Gold Q24 Reaktifleri</td><td>Qiagen</td><td>970802</td><td></td></tr><tr><td>PyroMark PCR kiti&nbsp;</td><td>Qiagen</td><td>978703</td><td></td></tr><tr><td>PyroMark Q24 MDx</td><td>Qiagen</td><td></td><td>Pyrosequencing çalışma istasyonu&nbsp;</td></tr><tr><td>PyroMark Q24 Yazılım&nbsp;</td><td>Qiagen</td><td>v2.0.8</td><td>Pyrosequencing yazılımı&nbsp;</td></tr><tr><td>S5&reg; Yüksek Isabetli DNA Polimeraz</td><td>NEB</td><td>M0491</td><td></td></tr><tr><td>QuantiTect Reverse Transkripsiyon Kiti</td><td>Qiagen</td><td>205311</td><td></td></tr><tr><td>QuantStudio Absolute Q MAP16 Plaka Kiti</td><td>Uygulamalı Biyosistemler</td><td>A52865</td><td>Dijital PCR sistemi&nbsp;</td></tr><tr><td>SYBR Green I Nukleik Asit Jel Boyama, DMSO'da 10.000x konsantre</td><td>Invitrogen</td><td>S7563</td><td></td></tr><tr><td>T4 DNA Ligazı</td><td>NEB</td><td>M0202</td><td></td></tr><tr><td>T4 Polinükleotid Kinaz</td><td>NEB</td><td>M0201</td><td></td></tr><tr><td>TRIzol Reaktifi</td><td>Invitrogen</td><td>15596026</td><td></td></tr></tbody></table>

k562 hücrelerinin epigenetik mühendisliği: dcas9-dnmt3a ve -hdac1 füzyon proteinleri kullanılarak stabil hedefli dna metilasyonu için Çift vektörlü epizomal strateji

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

K562 Hücrelerinin Epigenetik Mühendisliği: dCas9-DNMT3A ve -HDAC1 Füzyon Proteinleri Kullanılarak Stabil Hedefli DNA Metilasyonu için Çift Vektörlü Epizomal Strateji

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

Biyomühendislik

Burada bir episomuna göre yeniden programlama strateji ve histon deasetilaz inhibitörleri kullanılarak periferal kandan alınan insan uyarılmış pluripotent kök hücrelerin üretilmesi için bir protokol açıklar.

Epizomlar ve HDAC inhibitörleri kullanarak Blood Verimli iPS Hücre Üretimi

Fare embriyonik kök hücre kültürü çalışmalarının iki kimyasal tabanlı protokol ayrıntılı olarak açıklanmıştır. Bu yeni yöntem Tet-aracılı oksidasyon (vitamin C) teşvik ve DNA hypomethylation ve fare embriyonik kök hücreler pluripotent korumak için 5-metilsitozin (PD0325901 tarafından) de novo sentezi bastırmak sinerjik mekanizmaları kullanır.

Fare embriyonik kök hücre kullanarak MEK inhibitörü PD0325901 ve C vitamini genomik Hypomethylation korumak için bir alternatif kültür yöntemi

Memeli hücreleri kimyasal indüklenebilir DNA hydroxymethylation ve epigenetik remodeling için içine bir mühendislik Böl-TET2 enzim (elma şarabı) tanıtmak için detaylı protokolleri mevcuttur.

Kimyasal-indüklenebilir DNA Hydroxymethylation ve epigenetik Remodeling için bir mühendislik Split-TET2 enzim

Kromatin çevre düzenlenmesi uygun gen ekspresyonu için gerekli temel bir işlemdir. Burada, biz gen ekspresyonu kromatin değiştirme alımı yoluyla kontrol etmek için bir yöntem tarif gene özgü ve geri dönüşümlü bir şekilde makine.

Kimyasal epigenetik değiştiriciler ile hücresel makine yeniden yönlendirerek Gen transkripsiyonu bastırmak

DNA epigenome temsil düzenleme güçlü bir tedavi yaklaşımı hedef. Bu iletişim kuralı üretim, arıtma ve toplama tüm-içinde-bir lentiviral vektörlerin İnsan İndüklenmiş pluripotent kök hücre (hiPSC)'epigenome düzenleme uygulamaları için CRISPR-dCas9-DNMT3A transgene yataklık açıklar-nöronlar türetilmiş.

Pluripotent kök hücre kaynaklı hastalık modelleri lentiviral vektör platformu için Epigenome düzenleme araçları verimli bir şekilde teslim insan içine indüklenen

Burada, gen transfeksiyonuna veya retroviral vektörlere ihtiyaç duymadan, memeli pluripotent hücrelerini verimli bir şekilde üretmek için kimyasal epigenetik silme kullanımını mekanosensing ile ilgili ipuçlarıyla birleştiren bir yöntem sunuyoruz. Bu nedenle, bu strateji translasyonel tıp için umut vericidir ve kök hücre organoid teknolojisinde kayda değer bir ilerlemeyi temsil etmektedir.

Memeli Pluripotent Hücreleri Üretmek için Epigenetik Modifikasyonu ve Biyomekanik İpuçlarını Birleştiren İki Adımlı Bir Strateji

Burada, yüksek, uzun vadeli, kararlı, hedefe yönelik susturma verimliliğine ve düşük genom çapında, hedef dışı aktiviteye sahip tasarlanmış transkripsiyonel baskılayıcıların (ETR'ler) in vitro seçimi için bir protokol sunuyoruz. Bu iş akışı, aday ETR'lerin başlangıçtaki karmaşık repertuarını, terapötik olarak ilgili ortamlarda daha fazla değerlendirme için uygun olan kısa bir listeye indirgemeye olanak tanır.

In Vitro Hedeflenen Epigenetik Susturma için Tasarlanmış Transkripsiyonel Baskılayıcıların Seçimi

Hematopoetik kök hücre biyolojisini ve hematolojik maligniteleri daha iyi incelemek için hematopoetik kök ve progenitör hücrelerdeki (HSPC'ler) somatik mutasyonları sadık bir şekilde modellemek için yeni stratejiler gereklidir. Burada, CRISPR / Cas9 kullanımını ve çift rAAV donör transdüksiyonunu birleştirerek HSPC'lerde heterozigot fonksiyon kazancı mutasyonlarını modellemek için bir protokol açıklanmaktadır.

İnsan Hematopoetik Kök ve Progenitör Hücrelerinde Onkojenik Heterozigot Fonksiyon Kazancı Mutasyonlarının Mühendisliği

Protokol, plazmid DNA transfeksiyonu ve mRNA nükleofeksiyonu kullanılarak insan hücre hatlarında Kümelenmiş Düzenli Aralıklı Kısa Palindromik Tekrarlar (CRISPR) tabanlı epigenom düzenleme yöntemlerini açıklar.

Plazmid DNA Transfeksiyonu ve mRNA Nükleofeksiyon Dağıtımı Kullanılarak İnsan Hücrelerinde CRISPR Epigenom Düzenleme

Homoloji aracılı uç birleştirme (HMEJ) DNA onarım yolu yoluyla birincil insan T hücrelerinde büyük, multisistronik yapıların yüksek verimli hedefli knock-in'ini elde etmek için CRISPR / Cas9 teknolojisini kullanmak için ayrıntılı bir protokol sağlanmıştır. Bu cGMP'ye uyarlanabilir protokolle tasarlanan T hücreleri, mükemmel hücre genişlemesi, sitotoksisite ve sitokin üretimini korur.

Büyük DNA Şablonlarının Homoloji Aracılı Uç Birleştirme Hedefli Entegrasyonu Yoluyla Birincil İnsan T Hücrelerinin Viral Olmayan Mühendisliği

Keşfedin K562 hücrelerinde dCas9-DNMT3A ve dCas9-HDAC1 füzyon proteinleriyle hedefli DNA metilasyonu için çift vektörlü epizomal CRISPR stratejisi. Bu yöntem stabil epigenetik düzenleme sağlar.

K562 Hücrelerinin Epigenetik Mühendisliği: dCas9-DNMT3A ve -HDAC1 Füzyon Proteinleri Kullanılarak Stabil Hedefli DNA Metilasyonu için Çift Vektörlü Epizomal Strateji (Video) | JoVE Journal

K562 Hücrelerinin Epigenetik Mühendisliği: dCas9-DNMT3A ve -HDAC1 Füzyon Proteinleri Kullanılarak Stabil Hedefli DNA Metilasyonu için Çift Vektörlü Epizomal Strateji

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