Highly efficient tamoxifen-inducible Cre recombination in embryonic, larval and adult zebrafish

Abstract

The ability to inactivate gene function in an adult organism is essential for studies of biological processes such as regeneration and behavior. This is best achieved by engineering an allele which could be conditionally inactivated using Cre recombinase and subsequently inactivating gene function using a drug-inducible Cre recombinase. Several recent studies clearly demonstrate feasibility of engineering such conditional alleles in zebrafish. Meanwhile, achieving sufficient degree of recombination to induce complete loss of function has remained a major limitation. Herein we address this limitation by engineering a recombinant ubiquitin promoter ubb ^R consisting of the zebrafish ubiquitin promoter supplemented with an intronic enhancer from the carp beta-actin2 gene. Using phiC31-mediated targeted integration, we demonstrate that ubb ^R clearly outperforms both parental promoters as well as currently available ubiquitous CreER ^T2 driver lines at all embryonic and larval stages tested. Furthermore, the ubb ^R :CreER ^T2 driver line we generated enables near-complete inactivation of floxed alleles in adult zebrafish hearts. Finally, we demonstrate that our ubb ^R promoter retains high activity when integrated at other genomic loci, making it uniquely suitable for robust expression of transgenes at all stages of zebrafish ontogenesis.

Highlights

• Used targeted integration to directly compare different CreER ^T2 drivers

• Generated a ubiquitous ubb ^R :CreER ^T2 driver line capable of near-complete inactivation of floxed genes in adult zebrafish hearts

• Demonstrated that the recombinant ubb ^R promoter is suitable for robust transgene expression when integrated at different genomic loci