Abstract
To define cis-acting genetic elements responsible for cell-specific transcriptional regulation of the CC10 gene, DNA sequences spanning nucleotides -2338 to +49 of the rat CC10 gene were linked to a reporter gene coding for chloramphenicol acetyltransferase (CAT). In transient expression assays, CC10 sequences were capable of restricting CAT expression to a human lung adenocarcinoma cell line similar to pulmonary Clara cells. Transgenic mice harboring the hybrid RtCC10-CAT construct expressed high levels of CAT activity specifically within protein extracts of lung and trachea. Transcripts for the CAT reporter gene colocalized with those for the endogenous murine CC10 gene within the airways of transgenic mice. Functional analysis of deletion mutants identified stimulatory, inhibitory, and cell type-specific transcriptional regulatory elements. The results of gel retention and DNaseI protection assays suggest that a transcriptional stimulatory region located between -320 and -175, and a cell type-specific regulatory element located between -175 and +49, result from a series of protein-DNA interactions occurring at -220 to -205 and -128 to -86, respectively. Lung epithelial specific transcriptional regulatory elements described herein will be useful for expression of chimeric genes within epithelial cells lining the trachea, bronchi, and bronchioles of mice.
Highlights
To define cis-acting geneticelements responsible for the lung and uteroufsrats and rabbitrse,spectively
+49 of the rat CClo gene were linked to a reporter gene mRNA within the raltung, with low levels of uterine exprescoding for chloramphenicol acetyltransferase (CAT)
CClo sequences were capable of restricting CAT expression to a human lung adenocarcinoma cell line similar to pulmonary Clara cells
Summary
Cell Expression cycles with 30-s denaturation at 94 "C, 20-s annealing at 60 "C, and 3-min extension at 72 "C, followedby 38 cyclesat which the annealing temperature was raised to 68 "C. Labeled DNA (1ng) was incubated with 2 pgof nuclear extract and 1.0 pgof poly(dI.dC) in a buffer containing 12 mM HEPES (pH 7.9), 4 mM Tris (pH 8.0), 1 mM EDTA, 1mM DTT, 5 mM MgC12, and 150 mM KCl. Concentrations of MgC12 and poly(dI.dC) were empirically determined to minimize nonspecific protein-DNA interaction. DNA binding reactions were carried out in a mixture containing 10 mM Tris, pH 7.5, 0.5 mM dithiothreitol, 5 mM MgC12, 0.1 mM EDTA, 75 mM KCl, 0.2 mM PMSF, 12% glycerol, 200 pg/ml poly(dI.dC), approximately 1 ng of labeled DNA (1 X 10' cpm/pg), and 20-60pg of nuclear extract. Specific activities of CAT within tissue extracts were determined by quantitation of the percentage chloramphenicol acetylation using methods described above. CClo RNA-RNA hybrids were washed in 50% formamide, 2 X SSC
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