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Conservative Site-specific Recombination and Phase Variation

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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

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Ligand-Controlled Site-Specific Recombination in Zebrafish.

Avinash Chekuru¹, Veronika Kuscha¹, Stefan Hans¹

¹Technische Universität Dresden, Biotechnology Center and DFG-Center for Regenerative Therapies Dresden Cluster of Excellence, Tatzberg 47-49, 01307, Dresden, Germany.

Methods in Molecular Biology (Clifton, N.J.)

|August 18, 2017

Summary

This study provides practical protocols for testing CreER^T2 constructs in zebrafish, enabling conditional gene knockout and lineage tracing. These methods facilitate advanced genetic manipulation in both developing and adult zebrafish models.

Keywords:

CreERT2 Site-specific recombination Tamoxifen Zebrafish

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Published on: June 17, 2014

Area of Science:

Genetics
Molecular Biology
Zebrafish Model Systems

Background:

Cre-lox technology is crucial for precise genome manipulation, including conditional knockout alleles and lineage tracing.
Cre and CreER^T2 are efficient in zebrafish, but the availability of driver and effector lines is limited.
Advancements in genome editing tools like TALEN and CRISPR/Cas are increasing interest in conditional Cre/lox systems in zebrafish.

Purpose of the Study:

To outline practical experimental steps for transient functionality tests of CreER^T2 driver and effector constructs in zebrafish.
To introduce detailed protocols for inducing CreER^T2-mediated recombination in vivo in embryonic and adult zebrafish.

Main Methods:

Development of transient functionality assays for CreER^T2 driver constructs.
Establishment of protocols for inducing CreER^T2-mediated recombination in zebrafish embryos.
Establishment of protocols for inducing CreER^T2-mediated recombination in adult zebrafish.

Main Results:

Demonstrated practical methods for assessing CreER^T2 construct functionality in zebrafish.
Provided detailed protocols for successful in vivo recombination induction using CreER^T2 in zebrafish at various developmental stages.
Facilitated the application of conditional gene manipulation strategies in zebrafish research.

Conclusions:

The developed protocols enable efficient transient testing and in vivo application of CreER^T2-mediated recombination in zebrafish.
These methods will accelerate the generation and utilization of conditional knockout and lineage-tracing models in zebrafish.
The study addresses the need for robust Cre/lox system implementation in zebrafish, supporting advanced genetic studies.