Abstract
Although zebrafish is used to model human diseases through mutational and morpholino-based knockdown approaches, there are currently no robust transgenic knockdown tools. Here we investigate the knockdown efficiency of three synthetic miRNA-expressing backbones and show that these constructs can downregulate a sensor transgene with different degrees of potency. Using this approach, we reproduce spinal muscular atrophy (SMA) in zebrafish by targeting the smn1 gene. We also generate different transgenic lines, with severity and age of onset correlated to the level of smn1 inhibition, recapitulating for the first time the different forms of SMA in zebrafish. These lines are proof-of-concept that miRNA-based approaches can be used to generate potent heritable gene knockdown in zebrafish.
Highlights
Zebrafish is used to model human diseases through mutational and morpholino-based knockdown approaches, there are currently no robust transgenic knockdown tools
Spinal muscular atrophy (SMA) is an autosomal recessive human disease characterized by motoneuron loss, progressive muscle weakness and premature death[19]
To select an appropriate vector for both in vitro artificial miRNA synthesis for zebrafish transient experiments and the generation of miRNA-expressing constructs for transgenesis, we generated three different plasmids compatible with the Tol[2] kit components and containing minimal elements for RNA synthesis[29]
Summary
Zebrafish is used to model human diseases through mutational and morpholino-based knockdown approaches, there are currently no robust transgenic knockdown tools. We investigate the knockdown efficiency of three synthetic miRNA-expressing backbones and show that these constructs can downregulate a sensor transgene with different degrees of potency Using this approach, we reproduce spinal muscular atrophy (SMA) in zebrafish by targeting the smn[1] gene. The use of tissue-specific promoters in transgenes would allow spatiotemporal control and tracking via co-expression of fluorescent markers This approach would be of considerable potential, several studies suggest that it is difficult to design efficient synthetic miRNAs, and that it is difficult to generate transgenic lines presenting potent knockdown[11,18]. Several transgenic lines with less potent smn[1] reduction developed adult onset phenotypes, including abnormal swimming behaviour, scoliosis and considerable weight loss This is the first time that a zebrafish model offers the possibility to investigate SMA at later stages. This approach would be useful for the study of genes that are embryonically lethal in null alleles, haploinsufficient, when spatiotemporal inhibition is desired or when several genes need to be downregulated simultaneously, such as in models of polygenic disorders
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