Project description:Low transfection efficiency poses a significant challenge to experimental success, making the improvement of gene-carrying plasmid delivery and expression essential across various research fields. The stability of transfected vector DNA or mRNA significantly impacts transgene expression, as the innate immune system may recognize plasmid DNA as foreign and initiate a degradation response. Currently, it is unclear whether RNA transcribed from plasmids can activate RNA sensors, affecting transfection efficiency. This study employed RNA sequencing to analyze cellular responses to various circular and linear DNAs across five cell lines—HEK293T, HCT116, HeLa, L02, and NCM460—revealing that the innate immune response is a primary contributor to low transfection efficiency. Additionally, we used ChIP-seq to detect H3K27ac histone marks to explore whether the immune response triggered by plasmid transfection is regulated by epigenetic mechanisms. Finally, we performed ChIP assays for IRF7 and p-IRF3 in HeLa cells transfected with the linear pcDNA3.1-neo plasmid to demonstrate that these two transcription factors can indeed bind to the RTV genes.
Project description:Low transfection efficiency poses a significant challenge to experimental success, making the improvement of gene-carrying plasmid delivery and expression essential across various research fields. The stability of transfected vector DNA or mRNA significantly impacts transgene expression, as the innate immune system may recognize plasmid DNA as foreign and initiate a degradation response. Currently, it is unclear whether RNA transcribed from plasmids can activate RNA sensors, affecting transfection efficiency. This study employed RNA sequencing to analyze cellular responses to various circular and linear DNAs across five cell lines—HEK293T, HCT116, HeLa, L02, and NCM460—revealing that the innate immune response is a primary contributor to low transfection efficiency. Additionally, we used ChIP-seq to detect H3K27ac histone marks to explore whether the immune response triggered by plasmid transfection is regulated by epigenetic mechanisms. Finally, we performed ChIP assays for IRF7 and p-IRF3 in HeLa cells transfected with the linear pcDNA3.1-neo plasmid to demonstrate that these two transcription factors can indeed bind to the RTV genes.
Project description:RNA Seq datasets from WT or AAV-injected mice treated with 10 mg/kg risdiplam or solvent. RNA Seq datasets from plasmid-transfection HEK293T cells treated with 1uM risdiplam or DMSO.
Project description:This study observed that reporter protein from plasmid DNA transfection expression levels were dose-nonlinear after transfection in THP-1 cells caused by activation of DNA sensing. The expression levels of target genes were significantly upregulated after blocking the STING pathway. Through analysis of changes in the bulk and single cell RNA-seq of heterozygous cellular response to DNA transfection, we clarified the DNA sensing-induced innate immune response and apoptosis.
Project description:We analysed the genexpression of dental follicle cells (DFCs) after 3 days osteogenic differentiation with BMP2 after transfection with a DLX3 plasmid (pDLX3) and after transfection with an empty plasmid (pEV)
Project description:The STARR-seq plasmid library used for transfection was sequenced to enable input normalization. The sequencing library was generated with the NEBNext Ultra II FS DNA Library Prep Kit (New England Biolabs) and sequenced on the Illumina iSeq 100
Project description:To analyze the impact of Aire on gene expression profile in a model cell line, we used 293T cells and transfected them either with an Aire expression plasmid pCMV-Aire (where mAire is driven by CMV promoter) or with a control plasmid pCMV2B. Total RNA was extracted 48 hours post transfection, processed and used for gene expression profiling by Affymetrix. The data demonstrate that Aire has a very broad impact, effecting (upregulating and downregulating) hundreds of differents genes, however these genes differ dramatically from its targets in medullary epithelial cells. Keywords: transfection
