Comparative Studies of Various Artificial microRNA Expression Vectors for RNAi in Mammalian Cells

Abstract

Artificial microRNA (amiRNA) has recently become an important RNA interference (RNAi) technology for gene therapy and gene function studies. Here nine expression strategies were employed to construct plasmid vectors expressing amiRNA (amiR-Fluc) against firefly luciferase (Fluc). Our results indicate that all nine vectors can successfully produce mature amiR-Fluc and specifically suppress the expression of Fluc, although the RNAi efficiency in different mammalian cells displays obvious differences. Among these nine vectors, three can efficiently co-express DsRed reporter gene linked with amiR-Fluc cassette. Moreover, the recommended number of concatenated amiRNAs in a multi-amiRNA expression vector should not be more than four, and the relative position of an amiRNA in the multi-amiRNA expression vector has no apparent influence on its RNAi activity. In summary, all these results described here provide valuable information for the rational design and application of amiRNA expression vector.

The regulation of silkworm fibroin L chain production by miRNA-965 and miRNA-1926 in insect cells

“…Identification target genes that miRNAs regulated by miRNAs is important for understanding their specific biological functions. Experimental validation of targets is currently done through in vitro reporter assays [19,[22][23][24]. For example, luciferase or enhanced green fluorescent protein (EGFP) based reporter assay in cell, in which the entire 3'UTR of the putative target gene is cloned downstream of reporter gene, after the introduction of a miRNA to the cell, has been used for rapid detection of miRNA target interaction in vitro.…”

Multi‑target inhibition by four tandem shRNAs embedded in homo‑ or hetero‑miRNA backbones

“…Hetero-BB multiplexed shRNAmiRs are reconstituted based on authentic polycistronic miRNA clusters (4)(5)(6), as well as artificial tandem-arrayed miRNAs (7), and typically show additive antiviral properties compared to their mono-counterparts. However, homo-BB multiplexed shRNAmiRs commonly adopt the backbone of either miR30 (8)(9)(10)(11)(12)(13) or miR155 (14)(15)(16)(17) to accommodate the same or different shRNAs, targeting one or more genes, yielding inconsistent results regarding efficacy and stability of the homo-BB scaffold. Little is known regarding the impact of backbone selection on multi-shRNAmiR construction due to a lack of comparative studies of both hetero-BB and homo-BB.…”

“…Structural composition was based on several considerations. Firstly, it is known that more than four shRNAmiR cassettes in tandem result in less efficient knockdown when using C-terminal firefly luciferase (Fluc)-specific shRNAmiR as a functional readout (16). Therefore, a tetrameric approach was chosen.…”

High-efficiency Generation of Multiple Short Noncoding RNA in B-cells and B-cell-derived Extracellular Vesicles

“…In recent years, only few reports demonstrated the expression of multiple sncRNAs in cells using either a retrovirus or plasmid DNA, 13,14,15 but no attempts were made to assess the release of the sncRNAs in the extracellular compartment or their inclusion in EVs. The results show that B-cells transfected with plasmid DNA carrying the nucleotide sequence of multiple sncRNAs in tandem undergo the simultaneous biogenesis and secretion of multiple sncRNA, including their release and incorporation in EVs, at high efficiency and specifically.…”