Insertion of nuclear factor-kappaB binding sequence into plasmid DNA for increased transgene expression in colon carcinoma cells.
To increase plasmid DNA (pDNA)-based transgene expression, 5, 10 or 20 repeats of nuclear factor kappaB (NF-kappaB) binding sequences were inserted upstream of the cytomegalovirus (CMV) promoter region of a conventional pDNA encoding firefly luciferase (pCMV-Luc) to obtain pCMV-kappaB5-Luc, pCMV-kappaB10-Luc and pCMV-kappaB20-Luc. Murine carcinoma colon 26 cells, in which NF-kappaB was constitutively activated, were co-transfected with a firefly luciferase-expressing pDNA and a renilla luciferase-expressing pDNA having no NF-kappaB binding sequences using cationic liposomes. The expression efficiency of pCMV-kappaB(n)-Luc was evaluated using the ratio of the luciferase activities. Increasing numbers of NF-kappaB binding sequences significantly increased transgene expression. The expression was increased by NF-kappaB activators and the effects were marked with pDNA having many NF-kappaB binding sequences. These results indicate that insertion of NF-kappaB binding sequences into pDNA is an effective approach to increase transgene expression in cancer cells in which NF-kappaB is activated.
Thanaketpaisarn
Oranuch
O
Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Nishikawa
Makiya
M
Okabe
Takayuki
T
Yamashita
Fumiyoshi
F
Hashida
Mitsuru
M
D000818:Animals; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism*; D004251:DNA Transposable Elements / Q000235:genetics*; D051379:Mice; D016328:NF-kappa B / Q000235:genetics*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
D004251:DNA Transposable Elements; D016328:NF-kappa B