研究目的
To describe methods for using optogenetic heterodimerization to control protein localization and signaling in vertebrate embryos, specifically in zebrafish, using light to reversibly bind proteins and alter biological processes.
研究成果
The phytochrome optogenetic system enables precise, reversible control of protein localization and signaling in vertebrate embryos with high spatiotemporal resolution. It is particularly suited for long-term experiments in 3D tissues, though it requires careful handling to avoid background activation and relies on external chromophore addition. Future improvements may include genetic engineering for endogenous PCB synthesis and enhanced light patterning techniques.
研究不足
The phytochrome system requires an external chromophore (PCB) that must be added via microinjection, which adds complexity. Background dimerization can occur under certain light conditions, requiring careful control with unbinding light. The method is sensitive to light exposure, and precise balancing of PHYB and PIF RNA concentrations is critical. Spatial resolution is limited to specific z-planes without advanced patterning systems like DMD or SLM, and there may be heterogeneity in expression between cells.
1：Experimental Design and Method Selection:
The methodology involves using the phytochrome heterodimerization system for optogenetic control, adapted from plant systems for use in zebrafish embryos. It includes cloning plasmids, synthesizing mRNA, injecting into embryos, mounting for imaging, and patterning light with a confocal laser scanning microscope.
2：Sample Selection and Data Sources:
Early zebrafish embryos are used, with mRNA injected at the 8-64 cell stage for mosaic distribution. Samples are prepared and imaged live.
3：List of Experimental Equipment and Materials:
Includes plasmids (e.g., pCS2+ vector), restriction enzymes, PCR kits, electrophoresis equipment, mRNA synthesis kits, microinjection equipment (e.g., micropipette puller, micromanipulator), confocal microscope with specific lasers, external light sources (e.g., 740 nm LED), and chemicals like PCB chromophore.
4：Experimental Procedures and Operational Workflow:
Steps involve plasmid cloning using Gibson assembly, mRNA synthesis with NotI digestion, ethanol precipitation, RNA transcription and purification, embryo injection with RNA and PCB, mounting in low-melt agarose, and live imaging with patterned light illumination using ROI functions or external devices.
5：Data Analysis Methods:
Imaging data is analyzed to assess protein recruitment and localization changes, with adjustments based on pilot experiments for light intensity and timing.
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