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• W2896638602 abstract "ABSTRACT The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter A) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support canonical NF-κB signaling in response to treatment with tumor necrosis factor (TNF). Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic NEMO in the edited cells restores downstream NF-κB signaling in response to TNF. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter A) in the human SNU-423 liver cancer cell line. We have also used homology directed repair (HDR) to fix the promoter B element in a 293T cell clone. Overall, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed." @default.
• W2896638602 created "2018-10-26" @default.
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• W2896638602 date "2018-10-23" @default.
• W2896638602 modified "2023-09-26" @default.
• W2896638602 title "CRISPR/Cas9-based Editing of a Sensitive Transcriptional Regulatory Element to Achieve Cell Type-Specific Knockdown of the NEMO Scaffold Protein" @default.
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• W2896638602 doi "https://doi.org/10.1101/450320" @default.
• W2896638602 hasPublicationYear "2018" @default.
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