Figure 1.

Functional expression of Shh by administration of AdShh in vitro and in vivo in postnatal day 19 C57BL/6 mice. (a) Shh mRNA after in vitro infection of A549 epithelial cells with AdShh. A549 cells (10⁶) were infected (20 moi) with the AdNull control vector or AdShh. After 2 days, RNA was analyzed by Northern blot with a ³²P-labeled Shh cDNA probe. Uninfected cells (naive) and AdNull-infected cells were negative, whereas AdShh-infected cells contained a transcript of approximately 3.0 kb corresponding to the vector-encoded Shh transgene. Equal RNA loading was confirmed by analysis of GAPDH mRNA. (b) Shh protein expressed in vitro. Protein from A549 cells infected with AdNull or AdShh as described in a was analyzed by immunoblot for Shh protein. AdShh-infected, but not naive or AdNull-infected, A549 cells showed a 19-kDa immunoreactive band with anti-Shh. Equal protein loading was confirmed by Coomassie blue staining of an identically loaded gel (not shown). (c) Time course demonstrating expression of Shh, Ptc, and Gli1 mRNA in vivo. Dorsal skin was collected from naive C57BL/6 mice or C57BL/6 mice at postnatal days 19, 22, 24, 26, and 33 after intradermal injection on day 19 with AdNull or AdShh (10⁸ PFU for either vector). Skin was analyzed for Shh, Ptc, or Gli1 mRNA by Northern analysis on experimental days 0, 3, 5, 7, and 14. Expression of the 3.0-kb mRNA vector-encoded transcript (upper arrow) and the 2.6-kb endogenous Shh transcript (lower arrows) was detected in AdShh-injected mice, but the vector-derived transcript temporally preceded the endogenous transcript. Marked upregulation of Ptc and Gli1 gene expression was detected in AdShh-injected mice on experimental days 3 and 5, when low levels of expression of these genes were seen in naive and AdNull-treated mice. Equal RNA loading was confirmed by analysis of GAPDH mRNA.