Psilencer 4.1 cmv neo plasmid

The expression vectors for human OR3A4 and PDLIM2 were constructed using PCR methods. The PCR products were confirmed by direct DNA sequencing and cloned into the mammalian expression vectors pEGFP-N1 and pcDNA 3.0 as previously described [3 (link)]. The siRNA expression vector, the pSilencer 4.1-CMV/neo plasmid (Invitrogen Corp, Carlsbad, CA, USA), was used for cloning small synthetic oligonucleotides. Two sequences unique to the coding region of OR3A4 were designed and inserted between the BamHI and HindIII sites of the pSilencer 4.1-CMV/neo plasmid. The primers used to construct the three vectors are listed in Supplementary Table S2. MKN-45 and NCI-N87 gastric cancer cell lines were used for overexpression and knockdown studies. For transfection, complexes of Lipofectamine 2000 (Invitrogen) and the desired plasmid were prepared according to the manufacturer's instructions. We obtained stably transfected clones by G418 selection (Promega, Madison, WI, USA). The level of OR3A4 expression after transfection was assayed by RT-PCR. A stable transfectant of the empty vector was used as a control.

To construct a siRNA expression plasmid (P-422), a 21 nt siRNA sequence (5′-ATGGGCATTAATTGCATGCAT-3′) was designed at Whitehead (http://sirna.wi.mit.edu/) to target the SjAlstR gene. On the basis of this 21 nt target sequence, two complementary 55 nt siRNA template oligonucleotides were designed, synthesized, annealed and ligated into P-silencer 4.1-CMV neo plasmid (Ambion, USA). A control, scrambled sequence (5′-GCGAGTACCTTGTTAGATATA-3′) was cloned into P-silencer4.1-CMV neo to serve as a negative control (P-S422).
For each mouse at 20 d.p.i., a 1.5 ml transfection mixture, which contained 0.9% NaCl, 100 μg plasmid DNA and 150 μg PEI (AparnaBio, USA), was injected via tail vein. After maintenance of these mice for the subsequent 72 h, the mice were killed. The females in paired worms were collected for qRT–PCR investigation and for visual examination using light microscopy and CLSM. Mice that were not subjected to the injection of test compounds served as controls. Five independent experiments were performed.