Construction of an Artificial MicroRNA Expression Vector for Simultaneous Inhibition of Multiple Genes in Mammalian Cells

Abstract

Abstract:Recently, artificial microRNA (amiRNA) has become a promising RNA interference (RNAi) technology. Here, we describe a flexible and reliable method for constructing both single-and multi-amiRNA expression vectors. Two universal primers, together with two specific primers carrying the encoding sequence of amiRNA were designed and utilized to synthesize the functional amiRNA cassette through a one-step PCR. With appropriate restriction sites, the synthesized amiRNA cassettes can be cloned into any site of different destination vectors. Using the method, we constructed both singleand multi-amiRNA expression vectors to target three reporter genes, which code firefly luciferase (Fluc), enhanced green fluorescent protein (EGFP) and β-galactosidase (LacZ), respectively. The expressions of three genes were all specifically inhibited by either the corresponding single-or the multi-amiRNA expression vector in 293T cells. And the RNAi efficiency of each amiRNA produced by both single-and multi-amiRNA expression vectors was comparable.

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

“…The method for synthesizing amiR-Fluc, amiR-EGFP (amiRNA against the gene of enhanced green fluorescent protein), and amiR-LacZ (amiRNA against b-galactosidase reporter gene) cassettes was described in our previous report [26], and the construction of pDsRed (expressing red fluorescent protein), pDsRed-amiREGFP, pDsRed-amiRFluc, and multi-amiRNA expression vectors was also described [26]. With appropriate restriction sites, the synthesized amiR-Fluc cassette can be inserted into different destination vectors.…”

“…Here three amiRNA cassettes, which are against Fluc, EGFP, and lacZ reporter gene, respectively, were inserted into the pDsRed vector in different orders. The RNAi effects of these amiRNAs had been verified previously [26]. The position of amiR-Fluc cassette was changed from the first to the last in multiamiRNA expression vectors (Fig.…”

“…Total RNA extraction, electrophoresis, blotting, and hybridization were performed as previously described [26]. The amiR-Fluc was probed with a digoxigenin-labeled 21-nt DNA.…”

Survivin-targeting Artificial MicroRNAs Mediated by Adenovirus Suppress Tumor Activity in Cancer Cells and Xenograft Models

“… 15 , 16 , 17 Exogenous RNAi has been expressed in mammalian cells and animals in response to artificial microRNA (amiRNA) transcripts among a background of cellular miRNAs. 18 AmiRNAs are regarded as regulatory molecules by sequence-specific base pair that binds to the targeted mRNA, contributing to direct mRNA degradation or translational inhibition. 19 , 20 Owing to the specificity and efficiency in gene silencing, it has been increasingly investigated whether amiRNA targeted to oncogenes can correct aberrant transcript levels in cancer cells.…”

Advances in highly specific plant gene silencing by artificial miRNAs

“…Polycistronic amiRNA can target with many different genes, but whether the relative position of an amiRNA in the polycistronic pri-amiRNA transcript would affect its maturation and RNAi efficiency is the mainly concerned problem. The RNAi effects of these amiRNAs had been verified previously (Hu et al, 2009). Recently, Chen et al (2010) inserted three amiRNA cassettes, which are against Fluc, EGFP, and lacZ reporter gene respectively, into the pDsRed vector in different orders.…”