Tissue-specific and endogenous protein labeling with split fluorescent proteins.

Area of Science:

• Molecular Biology
• Genetics
• Zebrafish Model Systems

Background:

• Genetically encoded fluorescent proteins are vital for studying dynamic biological processes.
• Current methods for fluorescent protein fusion expression have limitations, particularly in whole organisms, including overexpression artifacts and technical difficulties with endogenous tagging.

Purpose of the Study:

• To overcome limitations of existing protein labeling techniques.
• To develop a method for tissue-specific and endogenous protein labeling in zebrafish using a split fluorescent protein system.

Main Methods:

• Utilized the split mNeonGreen2 (split-mNG2) system, comprising mNG2_1-10 and mNG2₁₁ fragments.
• Expressed mNG2_1-10 using tissue-specific promoters via standard transgenesis.
• Inserted mNG2₁₁ into endogenous genes of interest using CRISPR/Cas9 gene editing.
• Co-expressed both fragments to achieve self-assembly into a fluorescent complex.

Main Results:

• Successfully achieved differential labeling of cytoskeleton genes (tubb4b and krt8) in various zebrafish tissues using the split-mNG2 system.
• Demonstrated the ability to manipulate protein localization by anchoring the mNG2_1-10 component to specific cellular compartments.
• Confirmed that individual mNG2 fragments are non-fluorescent, with fluorescence only upon co-expression and assembly.

Conclusions:

• The split-mNG2 system provides a robust strategy for tissue-specific and endogenous protein labeling in zebrafish.
• This approach overcomes significant drawbacks of traditional protein fusion expression methods.
• The system offers broad utility for diverse applications in cell and developmental biology research.