Real-time imaging of gene expression in single living cells
نویسندگان
چکیده
منابع مشابه
Imaging real-time gene expression in living yeast.
INTRODUCTIONThis protocol describes the application of the MS2 system to the yeast Saccharomyces cerevisiae. ASH1 mRNA tagged with six MS2 repeats (6MBSs) is used to follow the localization of the ASH1 mRNA particles to the bud tip of a haploid yeast cell. W303 yeast cells transformed with pG14-MS2-GFP and pGAL-lacZ-MS2-ASH1 are grown on select medium lacking tryptophan and leucine. RNA express...
متن کاملImaging real-time gene expression in living systems with single-transcript resolution: image analysis of single mRNA transcripts.
INTRODUCTIONThis protocol describes a method for establishing a green fluorescent protein (GFP) calibration curve using dilutions of recombinant GFP and blue fluorescent beads. The total fluorescence intensity (TFI) per mRNA molecule is first calculated by imaging serial dilutions of purified enhanced GFP (eGFP) to determine the TFI within a specific volume. A calibration curve of fluorescence ...
متن کاملImaging real-time gene expression in living systems with single-transcript resolution: construct design and imaging system setup.
INTRODUCTIONThe most common way for a cell to respond to internal and external signals is to change its gene expression pattern. This requires the synchronization of regulatory steps along the expression pathway. Biological imaging techniques can be used to visualize and measure such processes in individual live cells in real time. This article discusses the use of a fluorescent RNA-binding pro...
متن کاملImaging Real-Time Gene Expression in Living Systems with Single-Transcript Resolution: Single mRNA Particle Tracking with ImageJ-Based Analysis.
INTRODUCTIONThis protocol describes the use of ImageJ software (freely available from NIH) to analyze particle dynamics in a cell using time-lapse movie frames or image stacks of fluorescent mRNA particles. Maximum intensity projections and kymographs are produced.
متن کاملImaging real-time gene expression in Mammalian cells with single-transcript resolution.
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 r...
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ژورنال
عنوان ژورنال: Chemistry & Biology
سال: 1998
ISSN: 1074-5521
DOI: 10.1016/s1074-5521(98)90287-3