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CRISPR/Cas9-mediated demethylation of FOXP3-TSDR toward Treg-characteristic programming of Jurkat T cells.
Wilk, Camilla; Effenberg, Laura; Abberger, Hanna; Steenpass, Laura; Hansen, Wiebke; Zeschnigk, Michael; Kirschning, Carsten; Buer, Jan; Kehrmann, Jan.
Afiliação
  • Wilk C; Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Germany.
  • Effenberg L; Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Germany.
  • Abberger H; Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Germany.
  • Steenpass L; Institute of Human Genetics, University Hospital Essen, University of Duisburg-Essen, Germany; Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany.
  • Hansen W; Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Germany.
  • Zeschnigk M; Institute of Human Genetics, University Hospital Essen, University of Duisburg-Essen, Germany.
  • Kirschning C; Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Germany.
  • Buer J; Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Germany.
  • Kehrmann J; Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Germany. Electronic address: jan.kehrmann@uk-essen.de.
Cell Immunol ; 371: 104471, 2022 01.
Article em En | MEDLINE | ID: mdl-34954490
ABSTRACT
Demethylation of FOXP3-TSDR (Treg specific demethylated region) is a hallmark of stable differentiation and suppressive function of regulatory T (Treg) cells. Previous protocols aiming at human naïve T cell differentiation failed to implement a Treg cell specific epigenetic signature. Ten-eleven translocation (TET) enzymes catalyze DNA demethylation. Plasmids towardexpression of a fusion protein encompassing nonfunctional Cas9, the catalytic domain of TET1, blue fluorescent protein, and encoding single guide RNAs (sgRNAs) targeting specific segments of the FOXP3-TSDR were engineered and transfected into Jurkat T cells. FOXP3-TSDR methylation was analyzed by deep-amplicon bisulfite sequencing while cellular Foxp3, Tbet, Gata3, and Rorgt mRNA levels were determined by real-time PCR. Overexpression of dCas9TET1 significantly decreased Jurkat cell FOXP3-TSDR methylation and increased Foxp3 mRNA expression while expressions of master transcription factor mRNAs of other major T cell lineages remained largely unaffected. dCas9-TET1 construct transfection mediated Treg programming of patients' primary T cells might be feasible.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Proteínas Proto-Oncogênicas / Linfócitos T Reguladores / Metilação de DNA / Fatores de Transcrição Forkhead / Sistemas CRISPR-Cas / Edição de Genes / Oxigenases de Função Mista Idioma: En Ano de publicação: 2022 Tipo de documento: Article País de afiliação: Alemanha

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Proteínas Proto-Oncogênicas / Linfócitos T Reguladores / Metilação de DNA / Fatores de Transcrição Forkhead / Sistemas CRISPR-Cas / Edição de Genes / Oxigenases de Função Mista Idioma: En Ano de publicação: 2022 Tipo de documento: Article País de afiliação: Alemanha