title: Identification of a minimal sequence of the mouse pro-alpha 1(I) collagen promoter that confers high-level osteoblast expression in transgenic mice and that binds a protein selectively present in osteoblasts.
abstract: Based on our previous transgenic mice results, which strongly suggested that separate cell-specific cis-acting elements of the mouse pro-alpha 1(I) collagen promoter control the activity of the gene in different type I collagen-producing cells, we attempted to delineate a short segment in this promoter that could direct high-level expression selectively in osteoblasts. By generating transgenic mice harboring various fragments of the promoter, we identified a 117-bp segment (-1656 to -1540) that is a minimal sequence able to confer high-level expression of a lacZ reporter gene selectively in osteoblasts when cloned upstream of the proximal 220-bp pro-alpha 1(I) promoter. This 220-bp promoter by itself was inactive in transgenic mice and unable to direct osteoblast-specific expression. The 117-bp enhancer segment contained two sequences that appeared to have different functions. The A sequence (-1656 to -1628) was required to obtain expression of the lacZ gene in osteoblasts, whereas the C sequence (-1575 to -1540) was essential to obtain consistent and high-level expression of the lacZ gene in osteoblasts. Gel shift assays showed that the A sequence bound a nuclear protein present only in osteoblastic cells. A mutation in the A segment that abolished the binding of this osteoblast-specific protein also abolished lacZ expression in osteoblasts of transgenic mice.
authors.affiliation: Department of Molecular Genetics, University of Texas, M. D. Anderson Cancer Center, Houston 77030, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Rossert
authors.forename: S S
authors.initials: SS
authors.lastname: Chen
authors.forename: H
authors.initials: H
authors.lastname: Eberspaecher
authors.forename: C N
authors.initials: CN
authors.lastname: Smith
authors.forename: B
authors.initials: B
authors.lastname: de Crombrugghe
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology; D004622:Embryo, Mammalian / Q000502:physiology; D004625:Embryo, Nonmammalian; D005314:Embryonic and Fetal Development; D015870:Gene Expression; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000096:biosynthesis; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010006:Osteoblasts / Q000378:metabolism*; D012516:Osteosarcoma; D011347:Procollagen / Q000096:biosynthesis* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000032:analysis / Q000096:biosynthesis
chemical_list: D011347:Procollagen; D008156:Luciferases; D001616:beta-Galactosidase