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

Fangfei Guo; Alisha Tromp; Haitao Wang; Thomas E Hall; Jean Giacomotto

doi:10.1093/nar/gkaf004

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

Journal article

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

Fangfei Guo, Alisha Tromp, Haitao Wang, Thomas E Hall and Jean Giacomotto

Nucleic Acids Research, Vol.53(2), pp.1-13

2025

DOI: https://doi.org/10.1093/nar/gkaf004

PMCID: PMC11744099

PMID: 39831309

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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.

Details

Title: Cre-Lox miRNA-delivery technology optimized for inducible microRNA and gene-silencing studies in zebrafish
Authors: Fangfei Guo - Griffith University
Alisha Tromp - The University of Queensland
Haitao Wang - The University of Queensland
Thomas E Hall - The University of Queensland
Jean Giacomotto (Corresponding Author) - University of the Sunshine Coast
Publication details: Nucleic Acids Research, Vol.53(2), pp.1-13
Publisher: Oxford University Press
Date published: 2025
DOI: 10.1093/nar/gkaf004
ISSN: 1362-4962
PMID: 39831309; PMC11744099
Copyright note: © The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Data Availability: The data underlying this article will be shared on reasonable request to the corresponding author.
Grants: ZebraClinics. From the identification of drugs against neurodegeneration to a better understanding of synaptic development and function., 1174145, National Health and Medical Research Council (Australia, Canberra) - NHMRC
Investigating the neuro-developmental role of schizophrenia-associated genes using the zebrafish, 1165850, National Health and Medical Research Council (Australia, Canberra) - NHMRC
Grant note: Rebecca L. Cooper Medical Research Foundation [PG2019405 to J.G.]; Cure SMA to J.G.
Organisation Unit: Thompson Institute
Language: English
Record Identifier: 991251393402621
Output Type: Journal article

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