ゼブラフィッシュにおけるCre/loxシステムを用いた遺伝子操作の進展

タイトル: Highly efficient tamoxifen-inducible Cre recombination in embryonic, larval and adult zebrafish

概要: We have generated transgenic lines containing zebrafish-optimized CreERT2 recombinase under the control of a recombinant ubbR promoter consisting of the zebrafish ubiquitin promoter supplemented with an intronic enhancer from the carp beta-actin2 gene. 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 promoter ubbR 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 ubbR clearly outperforms both parental promoters as well as currently available ubiquitous CreERT2 driver lines at all embryonic and larval stages tested. Furthermore, the ubbR:CreERT2driver line we generated enables near-complete inactivation of floxed alleles in adult zebrafish hearts. Finally, we demonstrate that our ubbRpromoter retains high activity when integrated at other genomic loci, making it uniquely suitable for robust expression of transgenes at all stages of zebrafish ontogenesis. HighlightsO_LIUsed targeted integration to directly compare different CreERT2 drivers C_LIO_LIGenerated a ubiquitous ubbR:CreERT2 driver line capable of near-complete inactivation of floxed genes in adult zebrafish hearts C_LIO_LIDemonstrated that the recombinant ubbR promoter is suitable for robust transgene expression when integrated at different genomic loci C_LI