Cre-Lox miRNA-delivery technology optimized for inducible microRNA and gene-silencing studies in zebrafish

Abstract

While many genetic tools exist for zebrafish, this animal model still lacks robust gene-silencing and microRNA-delivery technologies enabling spatio-temporal control and traceability. We have recently demonstrated that engineered pri-miR backbones can trigger stable gene knockdown and/or express microRNA(s) of choice in this organism. However, this miRNA-expressing technology presents important limitations. First, to trigger potent knockdown(s), multiple synthetic-miRNAs must be expressed simultaneously, compromising the co-expression of fluorescent marker(s) and knockdown traceability. Second, when gene(s) knockdown triggers significant phenotypes, like homozygous mutants with severe early phenotypes, it is difficult, if not impossible, to maintain transgenic carriers. To solve these problems and provide a mature RNAi and microRNA-delivery technology, we have generated new RNAi reagents and an inducible delivery system based on the Cre/Lox technology. This system allows the creation of asymptomatic/silent carriers, easing the production of embryos with potent knockdowns that can be traced and spatiotemporally controlled. We further demonstrated the utility of this approach by establishing novel inducible and tissue-specific models of spinal muscular atrophy, opening new avenues for studying smn1-gene function and pathogenicity. All in all, these materials and techniques will be invaluable in studying microRNA biology and in modelling or tackling conditions in which gene dosage is key.