Abstract
RNAi-based strategies have been used for hypomorphic analyses. However, there are technical challenges to achieve robust, reproducible knockdown effect. Here we examined the artificial microRNA (amiRNA) architectures that could provide higher knockdown efficiencies. Using transient and stable transfection assays in cells, we found that simple amiRNA-expression cassettes, that did not contain a marker gene (−MG), displayed higher amiRNA expression and more efficient knockdown than those that contained a marker gene (+MG). Further, we tested this phenomenon in vivo, by analyzing amiRNA-expressing mice that were produced by the pronuclear injection-based targeted transgenesis (PITT) method. While we observed significant silencing of the target gene (eGFP) in +MG hemizygous mice, obtaining −MG amiRNA expression mice, even hemizygotes, was difficult and the animals died perinatally. We obtained only mosaic mice having both “−MG amiRNA” cells and “amiRNA low-expression” cells but they exhibited growth retardation and cataracts, and they could not transmit the –MG amiRNA allele to the next generation. Furthermore, +MG amiRNA homozygotes could not be obtained. These results suggested that excessive amiRNAs transcribed by −MG expression cassettes cause deleterious effects in mice, and the amiRNA expression level in hemizygous +MG amiRNA mice is near the upper limit, where mice can develop normally. In conclusion, the PITT-(+MG amiRNA) system demonstrated here can generate knockdown mouse models that reliably express highest and tolerable levels of amiRNAs.
Highlights
RNA interference (RNAi)-mediated gene silencing is a useful approach for rapidly obtaining hypomorphic phenotypes by repressing selected gene
We examined the difference in the normalized mean fluorescence intensity (MFI) for eGFP and tdTomato across three AWVΔex lines by analysis of variance (ANOVA) in which the effects of line and experiment were included as the independent factor variables
To establish robust knockdown system, we first investigated the optimal structure for artificial microRNA (amiRNA) expression cassettes that could produce effective and desirable levels of gene knockdown
Summary
RNA interference (RNAi)-mediated gene silencing is a useful approach for rapidly obtaining hypomorphic phenotypes by repressing selected gene. Many different strategies are used for RNAi-mediated gene silencing that involve the expression of sequences such as short interference RNA (siRNA), short hairpin RNA (shRNA) or artificial microRNA (amiRNA) These sequences are designed to include the complementary sequence for a target mRNA with which they bind in RNA-induced silencing complex that will repress target gene expression (knockdown) both in vitro and in vivo [1, 2]. It is reported that high shRNA expression levels can result in severe toxicity in some tissues (e.g., liver, central nervous system, and heart) and/or lethality in mice, rats, and dogs [3, 7] These potential adverse effects of in vivo RNAi might be alleviated by using shRNA embedded within a natural miRNA backbone ( known as artificial microRNA/amiRNA) in vivo toxicity of amiRNA architectures is not well studied
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