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

Abstract

SummaryWe have generated transgenic lines containing zebrafish-optimized CreERT2recombinase under the control of a recombinantubbRpromoter consisting of the zebrafish ubiquitin promoter supplemented with an intronic enhancer from the carpbeta-actin2gene. These lines enable highly efficient tamoxifen-inducible recombination in embryonic, larval and adult zebrafish.AbstractThe 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 promoterubbRconsisting of the zebrafishubiquitinpromoter supplemented with an intronic enhancer from the carpbeta-actin2gene. Using phiC31-mediated targeted integration, we demonstrate thatubbRclearly outperforms both parental promoters as well as currently available ubiquitous CreERT2driver lines at all embryonic and larval stages tested. Furthermore, theubbR:CreERT2driver line we generated enables near-complete inactivation of floxed alleles in adult zebrafish hearts. Finally, we demonstrate that ourubbRpromoter retains high activity when integrated at other genomic loci, making it uniquely suitable for robust expression of transgenes at all stages of zebrafish ontogenesis.HighlightsUsed targeted integration to directly compare differentCreERT2driversGenerated a ubiquitousubbR:CreERT2driver line capable of near-complete inactivation of floxed genes in adult zebrafish heartsDemonstrated that the recombinantubbRpromoter is suitable for robust transgene expression when integrated at different genomic loci