Fig. 3.

Construction of the C-TSC cassette. a Expression of KLF16 (top) or REIC/Dkk-3 (bottom) proteins after transfecting the indicated constructs including a vector containing the CAG promoter in HEK293 cells. Tubulin was used as a control for loaded amounts of protein. b To further improve the expression, 3 promoter sequences of hTERT, SV40, and CMV were tandemly inserted downstream of the REIC cDNA-BGH (bovine growth hormone)-polyA signal. RU5′, a part of the HTLV type 1 LTR inserted for better efficiency of transcription and translation. CMV–CMV and CMV–RU5′–hTERT–SV40–CMV were constructed on the basis of pDNR-1r and pIDT-SMART promoter-less vectors (top). The vectors were transfected to HEK293 cells, and levels of inserted REIC protein were determined by Western blot analysis (top). Tubulin was used as a control for loaded amounts of protein. Finally, we named the improved high expression cassette C-TSC (bottom)