Pcdna 6.2 gw mir vector

Four potential miRNAs were developed from the complete IRES nucleotide sequence of FMDV O/HN/CHA/93 strain by using the miRNA design tool on Invitrogen’s web site tool (http://rnaidesigner.invitrogen.com/rnaiexpress/, Table 1). Oligonucleotides of the pre-miRNAs forward and reverse strands were synthesized, annealed, and cloned into pcDNA™6.2-GW/miR vector (Invitrogen) under the control of P_CMV and a transcriptional termination signal (TK pA), following the manufacturer’s protocol. These plasmids were designated pmiR153, pmiR220, pmiR242, and pmiR276 (Figure 1A). For subcloning, BamH I/Xho I digested products from pmiR276 were inserted into pmiR242 at its Bgl II/Xho I sites, resulting in pmiR242 + 276, a Dual-miRNA plasmid containing two IRES-specific miRNA hairpin structures (Figure 1A). Then, BamH I/Xho I fragments were digested from pmiR242 + 276 and cloned into pcDNA™6.2-GW/EmGFP-miR using a BLOCK-iT™ Pol II miR RNAi Expression Vector Kit with EmGFP (Invitrogen), to generate the recombinant plasmid pEGFP-miR242 + 276 expressing EGFP (Figure 1A). The pcDNA6.2-GW/miR-negative control plasmid (pmiR-NC) was provided by Invitrogen (Table 1) and has no sequence homology with FMDV. All of these plasmids were confirmed by DNA sequencing.

Various expression vectors were subcloned either to pcDNA3 (Invitrogen), pEGFPC (Clontech), or pcDNA6/BioEase (Invitrogen) using conventional molecular biology techniques and PCR^{14 (link),31 (link),32 (link)}. The sequence of Rab27a shRNA (5'-GCTGCCAATGGGACAAACATA-3') was used^{23 (link)} and inserted into pcDNA 6.2-GW/miR vector (Invitrogen).
In the Ras experiments, we used H-Ras expression vector set (Clontech, pCMV-Ras vector, and pCMV-RasV12 vectors). GFP-ubiquitin was a gift from Nico Dantuma (Addgene plasmid # 11928)^{33 (link)}.