Pentrtm1a vector

Full-length cDNA of Oip5 from mouse colon were subjected to RT-PCR by using Pfu DNA polymerase (Promega, Madison, WI) with primers containing a restriction enzyme (XhoI and KpnI) cutting site at the end. Primer sets were as follows: 5′-AGGGTACCACCATGGCGACTCTCTCGCGCCGCAG-3′ and 5′-CCGCTCGAGTTACAGGATCTTTGGTGATGCTGTTAACC-3′. The amplicons were cloned into pENTRTM1A vector (Life technology, Carlsbad, CA, USA) using restriction enzyme sites. After confirmation of correct sequences, the gene encoding Oip5 in pENTRTM1A vector was transferred into the adenoviral expression vector (pAd/CMV/V5-DEST; Life technology, Carlsbad, CA, USA) by recombination following the manufacturer’s instructions. The resultant pAd/CMV plasmid containing the target gene was linearized by PacI digestion and were transfected into 293A cells by lipofectamine-2000 (Life technology, Carlsbad, CA, USA). On day 2 after transfection, the 293A cells were passaged and were cultivated until 80% of cells became detached. The cell suspension was then frozen and thawed three times. After centrifugation at 1,750×g for 15 min, the supernatant was used as the gene expression adenoviral preparation (Ad-Oip5). The titers for the adenoviral preparation were around 5×10⁸ plaque forming units (pfu)/mL. The adenovirus expressing β-galactosidase (Ad-βgal) was used as a control.