A number of natural microRNA (miRNA) hairpins have been found in clusters of multiple identical or different copies, suggesting that effects of miRNAs can be enhanced and multiple genes can be regulated together by encoding multiple miRNA hairpins in a single transcript. Here, we report a simple and effective artificial multi-hairpin method that stimulates production of mature 22-nucleotide small RNAs from modified miRNA hairpins, improves gene knockdown over single-hairpin constructs, and provides linked multi-gene knockdowns.

Introduction

A recent advance in gene silencing methodology is to modify the stem sequence of miR-30 microRNA (miRNA) hairpin to achieve knockdown of artificially targeted genes (1,2,3,4,5,6,7). Modified miR-30 can achieve more effective knockdown than previous short-hairpin RNA (shRNA)-based methods (3,6) and can be expressed from pol II promoters. Nevertheless, knockdown efficiencies vary for different target genes or with different ways of expressing the modified miR-30, especially when knockdown vectors are present in low numbers in target cells (5,6,7).

A number of natural miRNA hairpins exist in clusters of multiple identical or different copies (8,9). This finding suggests that polycistronic transcripts might be naturally used to enhance the efficiencies of target gene repression or to achieve linked multi-gene repression. It is logical to hypothesize that polycistronic transcripts could be generated artificially to achieve better knockdown and linked multi-gene knockdown by modified miRNAs. In this report, we present a simple multi-hairpin design that effectively improves knockdown over single-hairpin constructs and provides linked multi-gene knockdowns.

Materials and Methods

Plasmid Construction

We followed a previous protocol (10), with modifications in PCR primer design, to construct the 118-nucleotide (nt) modified miR-30 hairpin (as shown in Figure 1A) flanked by artificial sequences containing various restriction sites (as shown in Figure 1B). PCR template and primer sequences are listed in Table 1.

Figure 1. An artificial multi-microRNA (miRNA) hairpin design. (Click to enlarge)

Table 1. Sequences of PCR Templates, Primers, and Hybridyzation Probes (Click to enlarge)

The 97-nt template was amplified with Vent_R® DNA polymerase (New England Biolabs, Ipswich, MA, USA) by 25 PCR cycles, each consisting of 30 s at 94°C, 30 s at 54°C, and 1 min at 75°C. Subsequently, 1 L Taq DNA polymerase was added to the PCR product and incubated at 72°C for 10 min. The PCR product was then cloned into pCR2.1-TOPO and confirmed by sequencing. Subsequent cloning steps are illustrated in Figure 1C. Single- or multi-hairpin constructs were inserted into XbaI and BamHI sites of lenti-CMV-GFP(11).

Cell Culture, Generation of Lentivirus, and Infection

293T cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Lentivirus were generated by cotransfecting 293T cells with various lentivirus plasmids and packaging plasmids (pMDLg/pRRE, pRSV-REV, and pMD2-VSVG) (11) by standard calcium phosphate protocol. After 48 h, supernatant was collected and filtered through a 0.2-µm filter. Virus titers were determined by infecting 293T cells with serial dilutions of virus stock and monitoring the percentage of green fluorescent protein (GFP) positive cells.

Northern Blot Analysis

Total RNA was extracted as described previously (8) and concentrated by precipitation with three volumes of ethanol. Thirty micrograms total RNA were fractionated on a 15% denaturing polyacrylamide gel, blotted in 0.5× Tris-borate-EDTA (TBE; 1.35 M Tris base, 0.45 M boric acid, 0.02 M EDTA) buffer onto a GeneScreen Plus® membrane (PerkinElmer Life and Analytical Sciences, Wellesley, MA, USA), and hybridized in ULTRAhyb®-Oligo buffer (Ambion, Austin, TX, USA). Ten femtomoles 19-nt antisense and sense oligonucleotides (Table 1) were also loaded to provide molecular weight and detection sensitivity references. The 19-nt sense oligonucleotides were end-labeled with T4 polynucleotide kinase (New England Biolabs). A U6 antisense oligonucleotide probe (Table 1) was used to probe for the 106-nt U6 RNA as control in the same Northern blot analysis. Total RNA was also fractionated on a 1.2% agarose/formaldehyde gel and blotted onto GeneScreen Plus membrane for analysis of large transcripts by hybridization in ULTRAhyb buffer to a randomly primed GFP or Skp2 cDNA fragment.

Western Blot Analysis

Cell lysates were prepared in lysis buffer [50 mM HEPES, pH 7.0, 250 mM NaCl, 0.1% Nonidet™ P40 (NP40), 5 mM EDTA, plus protease inhibitors]. Equal amounts of lysates (as determined by Bio-Rad Protein Assay Dye Reagent; Bio-Rad Laboratories, Hercules, CA, USA) were loaded onto a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Anti-Skp2 (Invitrogen, Carlsbad, CA, USA), anti-AR, anti-Cdk2, and anti-Cyclin A (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as primary antibodies. Horseradish peroxidase (HRP)-conjugated secondary antibodies were used for detection by chemiluminescence.