Knock-in tagging in zebrafish facilitated by insertion into non-coding regions

Area of Science:

• Zebrafish as a model organism
• Molecular biology
• Cell biology
• Genetics

Background:

• Zebrafish are valuable for in vivo cell biology due to live imaging capabilities.
• Traditional protein tagging via overexpression limits recapitulation of endogenous expression and function.
• Limitations in homologous recombination and targeted integration hinder endogenous tagging in zebrafish.

Purpose of the Study:

• To develop a simple and efficient method for endogenously tagging proteins in zebrafish.
• To enable accurate visualization and study of protein localization and function in vivo.
• To facilitate the generation of knock-in zebrafish lines for quantitative imaging.

Main Methods:

• Insertion of PCR-generated donor amplicons into non-coding gene regions.
• N- or C-terminal tagging of proteins with fluorescent proteins at endogenous loci.
• Generation of knock-in alleles for key genes involved in epithelial biology and organ development.

Main Results:

• Successfully generated endogenously tagged zebrafish alleles for ZO-1, Cldn15la, Rab11a, aPKC, and Integrin β1b.
• Demonstrated a simple approach for reliable protein tagging in zebrafish.
• Enabled accurate quantitative imaging studies through endogenously tagged proteins.

Conclusions:

• The developed method simplifies the generation of endogenously tagged zebrafish.
• This approach overcomes previous technical hurdles in zebrafish genetic modification.
• Facilitates advanced live imaging and functional studies of endogenous proteins in zebrafish.