Correct cell- and differentiation-specific expression of a murine alpha 1 (I) collagen minigene in vitro...

An in vitro differentiation system utilizing retinoic acid (RA) treatment of pluripotent murine P19 embryonal carcinoma (EC) cells, which can be induced to differentiate into various cell types, was optimized for maximal induction of alpha 1 type I collagen (Col1a1) gene expression. Differentiation was associated with apoptotic death of the majority of cells, indicating that this in vitro system faithfully mimics the in vivo differentiation process. Col1a1 mRNA became detectable by RNase protection assay after 3 days of RA treatment and, after 6 days, reached a level comparable to that in NIH 3T3 fibroblasts. After induction of differentiation the Col1a1 gene remained transcriptionally active for extended periods of time even in the absence of RA. A minigene version of the murine Col1a1 gene was constructed that contains all of the so far known Col1a1 regulatory elements. This construct exhibited the correct expression pattern in stable transfection experiments: it was expressed in fibroblasts, but not in undifferentiated P19 EC cells, and it was transcriptionally activated after induction of differentiation. This experimental system should be a useful tool for dissecting the molecular mechanisms involved in the developmental activation and stage- and tissue-specific expression of the murine Col1a1 gene.