Project description:Cell-penetrating peptides (CPP) uptake mechanism is still to be clarified to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by Pepfect 14, with or without oligonucleotide cargo on gene expression, on HeLa cells, have been investigated. The RNA expression was characterized by RNA sequencing.
Project description:We selected humann intervertebral disc samples to perform proteomics analysis. There were 1 case of grade I , 1 case of grade II, 3 cases of grade Ⅲ and 3 cases of grade Ⅳ according to Pfirrmann classfication. RNA seqencing analysis and single-cell RNA sequencing were integrated with proteomics data to identify the hub genes for intervertebral disc degeneration using bioinformatic method.
Project description:Response of HEK293-cells after transfection with EWS-FLI1. HEK293 cells were transfected with the expression vector pIRES2-EGFP containing type I EWS-FLI1 or empty control vector. For transient transfection standard DEAE dextran method was used and RNA was isolated 48h post transfection. For stable transfection cells were transfected using FuGENE 6 (Roche, Mannheim, Germany) and cells were selected with 400 ug/mL G418. DNA-microarray analysis was performed using Affymetrix HG-U133A microarrays.(see Staege et al. Cancer Res. 2004) Keywords = HEK293 Keywords = EWS-FLI1 Keywords = Ewing family tumors Keywords: other
Project description:We performed high-throughput sequencing of chromatin-associated RNA from CH12 cells depleted of Ddx1 and induced to undergo Class Switch Recombination (CSR).
Project description:Treatment of many pathologies of the brain could be improved markedly by the development of non-invasive therapeutic approaches that elicit robust, endothelial cell-selective, gene expression in specific brain regions that are targeted under MR image-guidance. While focused ultrasound (FUS) in conjunction with gas-filled microbubbles (MBs) has emerged as a non-invasive modality for MR image-guided gene delivery to the brain, it has been used exclusively to transiently disrupt the blood-brain barrier (BBB), which may induce a sterile inflammation response. Here, we introduce a new MR image-guided FUS method that elicits endothelial-selective transfection of the cerebral vasculature (i.e. “sonoselective” transfection), without opening the BBB. We first determined that activating circulating, cationic plasmid-bearing, MBs with pulsed low-pressure (0.1 MPa) 1.1 MHz FUS facilitates sonoselective gene delivery to the endothelium without MRI-detectable disruption of the BBB. The degree of endothelial selectivity varied inversely with the FUS pressure, with higher pressures (i.e. 0.3 MPa and 0.4 MPa FUS) consistently inducing BBB opening and extravascular transfection. Bulk RNA sequencing analyses revealed that the sonoselective low pressure regimen does not upregulate inflammatory or immune responses. Single cell RNA sequencing indicated that the transcriptome of sonoselectively transfected brain endothelium was unaffected by the treatment. The approach developed here permits targeted gene delivery to blood vessels and could be used to promote angiogenesis, release endothelial cell-secreted factors to stimulate nerve regrowth, or recruit neural stem cells.
Project description:Treatment of many pathologies of the brain could be improved markedly by the development of non-invasive therapeutic approaches that elicit robust, endothelial cell-selective, gene expression in specific brain regions that are targeted under MR image-guidance. While focused ultrasound (FUS) in conjunction with gas-filled microbubbles (MBs) has emerged as a non-invasive modality for MR image-guided gene delivery to the brain, it has been used exclusively to transiently disrupt the blood-brain barrier (BBB), which may induce a sterile inflammation response. Here, we introduce a new MR image-guided FUS method that elicits endothelial-selective transfection of the cerebral vasculature (i.e. “sonoselective” transfection), without opening the BBB. We first determined that activating circulating, cationic plasmid-bearing, MBs with pulsed low-pressure (0.1 MPa) 1.1 MHz FUS facilitates sonoselective gene delivery to the endothelium without MRI-detectable disruption of the BBB. The degree of endothelial selectivity varied inversely with the FUS pressure, with higher pressures (i.e. 0.3 MPa and 0.4 MPa FUS) consistently inducing BBB opening and extravascular transfection. Bulk RNA sequencing analyses revealed that the sonoselective low pressure regimen does not upregulate inflammatory or immune responses. Single cell RNA sequencing indicated that the transcriptome of sonoselectively transfected brain endothelium was unaffected by the treatment. The approach developed here permits targeted gene delivery to blood vessels and could be used to promote angiogenesis, release endothelial cell-secreted factors to stimulate nerve regrowth, or recruit neural stem cells.
Project description:Giardia lamblia is an important causative agent of persistent diarrhea in humans, domestic animals, and cattle. Basic research is usually performed with the strain WBC6 and includes genetic manipulations such as transfections. Here, we investigate how transfection with a plasmid causing stable expression of a foreign gene affects the whole proteome pattern. Using shotgun mass spectrometry, we compare the proteomes of untransfected trophozoites to trophozoites transfected with Escherichia coli glucuronidase A (GusA). Besides GusA, which is detected in the transfected trophozoites only, the proteomes of untransfected and transfected trophozoites differ by 132 differentially expressed proteins. In particular, transfection induces antigenic variation. Since transfection causing stable expression affects the proteome pattern, transfection experiments should take into account this effect. Due to a unique peptide panel, GusA is an example for a suitable internal standard for experiments involving transfected cells.
Project description:Total RNA was isolated from HuH-7 cells after transfection of IGF-II specific siRNAs. Gene expression profiling was performed using the Affymetrix Human Genome U133A 2.0 Arrays. The raw data were analysed using mixed model ANOVA. Keywords: transfection with siRNA