Temporally-controlled site-specific recombination in zebrafish

Area of Science:

• Genetics and Genomics
• Developmental Biology
• Molecular Biology

Background:

• The Cre-lox site-specific recombination system is widely used in mouse models but underutilized in other vertebrates.
• Previous attempts to use Cre-lox in zebrafish showed very low recombination efficiency, limiting its application.
• A need exists for efficient and controllable genetic tools in zebrafish for developmental and adult studies.

Purpose of the Study:

• To establish a highly efficient Cre-lox recombination system in zebrafish.
• To achieve temporal control over site-specific recombination using a ligand-inducible Cre variant.
• To demonstrate the utility of this system for mosaic labeling and studies in both embryonic and adult zebrafish.

Main Methods:

• Utilized transposon-mediated transgenesis to deliver Cre recombinase constructs into zebrafish.
• Employed the ligand-inducible CreER(T2) variant for temporal control of recombination.
• Administered tamoxifen (TAM) or 4-hydroxy-tamoxifen (4-OHT) to induce recombination and assessed kinetics and efficiency.

Main Results:

• Transposon-mediated delivery resulted in highly efficient Cre-mediated recombination in zebrafish, overcoming previous limitations.
• CreER(T2) mediated tamoxifen-inducible recombination with rapid kinetics, detectable within 2-4 hours of drug administration.
• Low doses of tamoxifen enabled mosaic labeling of single cells, providing precise spatial and temporal control.

Conclusions:

• Conditional Cre/lox systems are now a valuable and efficient tool for zebrafish research, applicable to both embryonic and adult stages.
• The developed method, utilizing single-copy insertion transgenesis, offers a robust strategy for conditional gene manipulation in zebrafish.
• This approach holds potential for adaptation to other vertebrate model organisms, expanding the utility of Cre-lox technology.