We designed the following oligonucleotides and plasmids according to the method of Koibuchi et al.29 (link) F2 (chick lysozyme TRE at nucleotides −2358 to −2326; half-sites arranged as an inverted palindrome with nucleotide gap of 6 (ref. 35 (link))); palindromic (typical TREs, which designed not to contain a putative RORE); DR4-1 (DR4 sequence containing AT-rich sequences at the 5′ end of the upstream half-site to serve as a putative RORE); and DR4-2 (DR4 oligonucleotide lacking an AT-rich sequence).36 (link) These oligonucleotides were cloned in the psi-CHECK2 vector (Thermo Fisher Scientific, USA) containing a viral thymidine kinase promoter coupled to the firefly luciferase-labelled gene which was used as the internal reference to remove the transfection efficiency differences between groups, and the Renilla luciferase reporter gene.37 (link) These reporter plasmids were sequenced to ensure that only a single copy of the TRE had been incorporated (Fig. 2A).
Accelrys DS Gene software (Accelrys Software, Inc., U.S.A.) was adopted to analyze the enzyme-cutting site of the psiCHECK2 promoter between the Poly A and hsv-tk promoter. The Bbs I enzyme was selected and the cutting site is shown in Fig. 2B. The nucleotide sequences of the double-stranded oligonucleotides containing TRE or RORE are shown in Fig. 2C.