Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency(1), potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.
Genes, Reporter , Luciferases/genetics , Macrophages/physiology , Transfection/methods , Animals , Cell Line , Mice , Plasmids/genetics
The anti-inflammatory cytokine interleukin-10 (IL-10) is essential for attenuating the inflammatory response, which includes reducing the expression of pro-inflammatory microRNA-155 (miR-155) in lipopolysaccharide (LPS) activated macrophages. miR-155 enhances the expression of pro-inflammatory cytokines such as TNFα and suppresses expression of anti-inflammatory molecules such as SOCS1. Therefore, we examined the mechanism by which IL-10 inhibits miR-155. We found that IL-10 treatment did not affect the transcription of the miR-155 host gene nor the nuclear export of pre-miR-155, but rather destabilized both pri-miR-155 and pre-miR-155 transcripts, as well as interfered with the final maturation of miR-155. This inhibitory effect of IL-10 on miR-155 expression involved the contribution of both the STAT3 transcription factor and the phosphoinositol phosphatase SHIP1. This is the first report showing evidence that IL-10 regulates miRNA expression post-transcriptionally.
Interleukin-10/pharmacology , Macrophages/drug effects , Macrophages/metabolism , MicroRNAs/genetics , RNA Precursors/genetics , RNA Processing, Post-Transcriptional/drug effects , RNA Stability/drug effects , Animals , Biological Transport , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Female , Gene Expression Regulation/drug effects , Inositol Polyphosphate 5-Phosphatases , Lipopolysaccharides/immunology , Macrophages/immunology , Male , Mice , MicroRNAs/metabolism , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphoric Monoester Hydrolases/metabolism , RNA Precursors/metabolism , STAT3 Transcription Factor/metabolism
The axon initial segment (AIS) contains the site of action potential initiation and plays a major role in neuronal excitability. AIS function relies on high concentrations of different ion channels and complex regulatory mechanisms that orchestrate molecular microarchitecture. We review recent evidence that a large number of ion channels associated with epilepsy are enriched at the AIS, making it a 'hotspot' for epileptogenesis. Furthermore, we present novel data on the clustering of GABRgamma2 receptors in the AIS of cortical and hippocampal neurons in a knock in mouse model of a human genetic epilepsy. This article highlights the molecular coincidence of epilepsy mutations at the AIS and reviews pathogenic mechanisms converging at the AIS.
Axons/physiology , Epilepsy/physiopathology , Ion Channels/physiology , Action Potentials/physiology , Adenoviridae/genetics , Animals , Axons/chemistry , Data Interpretation, Statistical , Electrophysiology , Gene Transfer Techniques , Humans , Ion Channels/genetics , Microscopy, Confocal , Receptors, GABA-A/genetics , Receptors, GABA-A/physiology , Sodium Channels/genetics , Sodium Channels/physiology , Tissue Fixation
