The CCT promoter directs high-level transgene expression in distal lung epithelial cell lines.
Gene therapy requires the presence of a robust and yet small promoter to drive high-level expression of desired proteins. In comparative analysis, we investigated the promoter strength of the CTP:phosphocholine cytidylyltransferase promoter (CCT alpha) with other commonly used promoters, which were all cloned into a similar background vector (PGL3 basic). Transient promoter-reporter assays in murine lung epithelial (MLE-12) cells revealed that the core CCT alpha promoter (240 bp) was observed to exhibit a 40-fold, 8-fold, and 3-fold higher level of activity compared with the simian virus 40, human cytomegalovirus, and Rous sarcoma virus promoters, respectively. The CCT alpha promoter was significantly more active than the Clara cell 10, thymidine kinase, and phosphoglycerate kinase promoters. This pattern of high-level expression for CCT alpha was detected primarily in cell lines of distal lung epithelial origin (MLE-12, RLE, H441) and was reduced in other cell lines (A549, CHO, HepG 2). CCT alpha promoter-reporter activity, CCT alpha transcript levels, and immunoreactive protein levels increased significantly in the presence of all-trans retinoic acid. The CCT alpha promoter, in a retinoic acid-inducible manner, efficiently directed expression of murine erythropoietin in MLE-12 cells. Collectively, these observations suggest that the CCT alpha construct might be useful to drive high-level, regulatable expression of heterologous proteins in alveolar epithelia.
Zhou
Jiming
J
Pulmonary Division, C-33K, GH, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242, USA. rama-mallampalli@uiowa.edu
You
Yong
Y
Zabner
Joseph
J
Ryan
Alan J
AJ
Mallampalli
Rama K
RK
D000818:Animals; D002460:Cell Line; D019916:Choline-Phosphate Cytidylyltransferase / Q000235:genetics*; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008168:Lung / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D019076:Transgenes
D019916:Choline-Phosphate Cytidylyltransferase