Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155

Abstract

Vector-based RNA interference (RNAi) has emerged as a valuable tool for analysis of gene function. We have developed new RNA polymerase II expression vectors for RNAi, designated SIBR vectors, based upon the non-coding RNA BIC. BIC contains the miR-155 microRNA (miRNA) precursor, and we find that expression of a short region of the third exon of mouse BIC is sufficient to produce miR-155 in mammalian cells. The SIBR vectors use a modified miR-155 precursor stem–loop and flanking BIC sequences to express synthetic miRNAs complementary to target RNAs. Like RNA polymerase III driven short hairpin RNA vectors, the SIBR vectors efficiently reduce target mRNA and protein expression. The synthetic miRNAs can be expressed from an intron, allowing coexpression of a marker or other protein with the miRNAs. In addition, intronic expression of a synthetic miRNA from a two intron vector enhances RNAi. A SIBR vector can express two different miRNAs from a single transcript for effective inhibition of two different target mRNAs. Furthermore, at least eight tandem copies of a synthetic miRNA can be expressed in a polycistronic transcript to increase the inhibition of a target RNA. The SIBR vectors are flexible tools for a variety of RNAi applications.

Activity-dependent BDNF release via endocytic pathways is regulated by synaptotagmin-6 and complexin

“…When DIV 5-6 hippocampal neurons were transfected with a micro-RNA-based siRNA vector (35), siCpx, together with vector expressing PSD95-GFP, we found that immunostaining of endogenous Cpx1/2 was down-regulated efficiently in these neurons on 13-15 DIV, but PSD95-GFP puncta were similar to that in control untransfected cells (Fig. 7 A and B).…”

Protogenin Defines a Transition Stage during Embryonic Neurogenesis and Prevents Precocious Neuronal Differentiation

“…The extracellular domain of PRTG and ERdj3 were then cloned into psFc after PCR as pPRTGe-Fc and pERdj3-Fc. Protocols described by Chung et al (2006) were followed to construct the microRNA mimics. DNA fragments containing PRTG or ERdj3 sequences and scramble sequences (supplemental Table S1, available at www.jneurosci.org as supplemental material) were cloned into vectors UI4 -puro-SIBR or UI4 -GFP-SIBR to give shPRTG(m), shERdj3(m), shPRTG(g), shERdj3(g), and shS [scrambled short-hairpin RNA (shRNA)].…”

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

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“…Northern blot analysis was performed as described previously [53] using 15µg of RNA per sample. Images were obtained with IPLab Gel H 1.5g software (Signal Analytics) from the PhosphorImager 445 SI (Molecular Dynamics) and were assembled with ImageJ software [57].…”