Abstract
The maturation of primary rat tracheal epithelial (RTE) cells seeded in denuded rat tracheas has been extensively studied. We are interested in using this system to study the expression of transgenes within differentiating cells of this regenerating epithelium. A focus of this study was to optimize promoter/reporter constructs for high levels of expression within the various cell types of the rat trachea. To this end we produced recombinant retroviruses from three β-galactosidase (β-gal) retroviral vectors that contained various promoters. The retroviruses were used to transduce the β-gal gene into primary cultures of RTE cells. These tagged cells were transplanted into denuded rat tracheas and grafted into nu/nu balb c mice. Following harvesting at 9, 21, and 42 days, the tracheas were analyzed immunohis-tochemically with anti-β-gal and histochemically with X-gal for β-gal-positive cells. Tracheas repopulated with 2 × 105 cells give rise to large undifferentiated clones at 9 days (some >500 cells) as determined by X-gal staining. The large size of these clones suggests that a small subpopulation of β-gal tagged primary cells are capable of rapid proliferation and repopulation of the tracheal grafts. Following 21 days grafting, differentiation of the regenerating epithelium was evident by the appearance of ciliated cells. By altering the promoter driving the β-gal gene, we were able to define which promoters were capable of high levels of expression within these differentiated ciliated cells. A fully differentiated epithelium harboring the β-gal marker gene was present by 6 weeks postgrafting.
Published Version
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