研究目的
To develop an in vitro culture system for real-time analysis of segmentation clock oscillations within single, isolated presomitic mesoderm (PSM) cells of zebrafish, enabling direct manipulation of the segmentation clock with single-cell resolution.
研究成果
The developed protocol provides a tool to directly study the cell autonomous nature of genetic oscillations in the segmentation clock in a quantitative way, enabling the measurement of dynamics of the oscillations in hundreds of cells under a variety of conditions. This method opens the door to a rigorous evaluation of factors that promote or inhibit oscillations in these cells.
研究不足
The observed period of single cells in culture is longer than the somitogenesis period in the intact embryo, and a variable period and amplitude is observed in most single-cell time series. The source of this variability is unknown.
1:Experimental Design and Method Selection:
The protocol involves generating dispersed cell cultures from zebrafish PSM tissue for time-lapse imaging of fluorescence signal from individual clock cells.
2:Sample Selection and Data Sources:
Transgenic zebrafish embryos heterozygous for the her1 fluorescent reporter allele are used.
3:List of Experimental Equipment and Materials:
Includes fire-polished glass pipette, fine forceps, Sylgard-coated 35-mm dish, tungsten wire tools, micro-scalpel, L15 medium with serum, trypsin/EDTA solution, Sigmacoted gel-loading tips, and Fibronectin1-coated glass-bottom dishes.
4:Experimental Procedures and Operational Workflow:
Dissection of PSM tissue from embryos, dispersal of cells, and time-lapse imaging setup.
5:Data Analysis Methods:
Image processing and analysis using Fiji software to track and measure fluorescence intensity over time in single cells.
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