CRISPR/Cas9-mediated demethylation of FOXP3-TSDR toward Treg-characteristic programming of Jurkat T cells.
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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Base de dados:
MEDLINE
Assunto principal:
Proteínas Proto-Oncogênicas
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Linfócitos T Reguladores
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Metilação de DNA
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Fatores de Transcrição Forkhead
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Sistemas CRISPR-Cas
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Edição de Genes
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Oxigenases de Função Mista
Idioma:
En
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Alemanha