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 single- and 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.
Highlights
MicroRNAs are endogenously encoded ~ 22 nt RNAs that can cause target mRNA degradation or translation repression, and play important roles in differentiation, development, cancer, or viral infections [1]
Three reporter genes encoding for firefly luciferase (Fluc), enhanced green fluorescent protein (EGFP) and LacZ were selected as targets, and the backbone of miR-155 was employed for the expression of artificial microRNAs (amiRNAs)
All amiRNA expression vectors constructed in the study were functionally effective, and the expressions of all three reporter genes were suppressed by either corresponding single- or multi-amiRNA expression vector. These results shows that multiple amiRNAs can be co-expressed from a single transcript, and RNA interference (RNAi) efficiency of each is comparable to that produced by the single amiRNA expression vector
Summary
MicroRNAs (miRNAs) are endogenously encoded ~ 22 nt RNAs that can cause target mRNA degradation or translation repression, and play important roles in differentiation, development, cancer, or viral infections [1]. Duplex sequence is replaced by the artificial one Such a design allows amiRNAs to be processed in a similar biogenesis pathway of natural miRNA and results in functional mature amiRNAs. Zeng and colleagues first successfully utilized miR-30 backbone to produce functional amiRNAs and suppress the expression of endogenous human genes [13]. Current methods for constructing amiRNA expression constructs require a special vector carrying the flanking sequence of natural miRNA or the synthesis of long oligonucleotides The former, such as the pCMV-miR-30 [19], pENTR/CMV-EGFP-miR-30 [20] or SIBR [21] vectors, lack flexibility for employing various miRNA backbones, using different promoters and generating multiamiRNA expression constructs. By either corresponding single- or multi-amiRNA expression vector, and the RNAi efficiency of the multi-amiRNA expression vector is comparable to that of the single one
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