Imaging real-time gene expression in Mammalian cells with single-transcript resolution.
ABSTRACT INTRODUCTIONThe MS2 system provides optimal sensitivity for single-molecule detection in cells. It requires two genetically encoded moieties: a reporter mRNA that contains MS2 binding site (MBS) stem loops and a fluorescent MS2 coat protein (MCP-xFP) that binds to the stem loops with high affinity, thus tagging the mRNA within the cell. This protocol describes transfection of COS-7 cells with reporter RNA (e.g., pRSV-Z-24 MBS-β-actin) and MCP-xFP (e.g., pPolII-MCP-GFP-NLS) plasmids using calcium phosphate precipitation. The reporter mRNA plasmid must be co-transfected with the MCP-xFP-NLS plasmid for simultaneous expression in a cell. The unbound MCP-xFP-NLS is sequestered in the nucleus, leaving only the MCP-xFP-NLS that is bound to the reporter mRNA in the cytoplasm. This provides a high signal-to-noise ratio (SNR) that permits detection of single mRNA molecules. The Delta T Imaging System is used for image acquisition of fluorescent particles in the cells.
- SourceAvailable from: Yousef Haj-Ahmad[Show abstract] [Hide abstract]
ABSTRACT: Plasmid DNA is widely used to deliver genes into mammalian cells for the construction of new cell lines, gene therapy and gene expression studies. During DNA preparation, various contaminants can be introduced and reduce its delivery efficiency and create mutations that decrease the expression level of the delivered genes. We evaluated the effect of different plasmid DNA contaminants on calcium phosphate-based transfection efficiency as well as gene expression in Chinese hamster ovary cells. pCMVβ was transfected into the cells after spiking with five different contaminants conditions: ethanol, endotoxin, cesium chloride, ethidium bromide and the combination of the latter two contaminants. Transfection efficiencies were determined through the counting of the Lac-Z-expressing cells as well as quantitative PCR. The reversibility of the contaminant interaction with DNA was examined through cleaning of the spiked DNA followed by transfection. As concluded from our results, qPCR offers accurate measurement of transfection efficiency than the conventional Lac-Z activity assay. In general, all of the examined contaminants, with the exception of ethanol, have a significant negative effect on gene expression. While this effect is resulted from the reduced delivery in most of the used contaminants, ethidium bromide showed no significant decrease in delivery, indicating alternative mechanism for its negative effect on gene expression.