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Published ahead of print on June 26, 2003, doi:10.1165/rcmb.2003-0020OC

Am. J. Respir. Cell Mol. Biol., Volume 30, Number 1, January 2004, 61-68

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Submitted on January 17, 2003
Revised on June 18, 2003

The CCT Promoter Directs High-Level Transgene Expression in Distal Lung Epithelial Cell Lines

Jiming Zhou1, Yong You1, Joseph Zabner1, Alan J Ryan2, and Rama K Mallampalli3*

1 Internal Medicine, University of Iowa, Iowa City, IA, USA, 2 Internal Medicine, University of Iowa, Iowa City, IA, USA; VA Medical Center, Iowa City, IA, USA, 3 Internal Medicine, University of Iowa, Iowa City, IA, USA; VA Medical Center, Iowa City, IA, USA; Biochemistry, University of Iowa, Iowa City, IA, USA

* To whom correspondence should be addressed. E-mail: rama-mallampalli{at}uiowa.edu.

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 (240bp) was observed to exhibit a 40-fold, 8-fold, and 3-fold higher level of activity compared to 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 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.




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