title: Expression of human smooth muscle calponin in transgenic mice revealed with a bacterial artificial chromosome.
abstract: Defining regulatory elements governing cell-restricted gene expression can be difficult because cis-elements may reside tens of kilobases away from start site(s) of transcription. Artificial chromosomes, which harbor hundreds of kilobases of genomic DNA, preserve a large sequence landscape containing most, if not all, regulatory elements controlling the expression of a particular gene. Here, we report on the use of a bacterial artificial chromosome (BAC) to begin understanding the in vivo regulation of smooth muscle calponin (SM-Calp). Long and accurate polymerase chain reaction, sequencing, and in silico analyses facilitated the complete sequence annotation of a BAC harboring human SM-Calp (hSM-Calp). RNase protection, in situ hybridization, Western blotting, and immunohistochemistry assays showed the BAC clone faithfully expressed hSM-Calp in both cultured cells and transgenic mice. Moreover, expression of hSM-Calp mirrored that of endogenous mouse SM-Calp suggesting that all cis-regulatory elements governing hSM-Calp expression in vivo were contained within the BAC. These BAC mice represent a new model system in which to systematically assess regulatory elements governing SM-Calp transcription in vivo.
authors.affiliation: Center for Cardiovascular Research, University of Rochester Medical Center, Rochester, New York 14642, USA. Joseph_Miano@urmc.rochester.edu
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Miano
authors.forename: Chad M
authors.initials: CM
authors.lastname: Kitchen
authors.forename: Jiyuan
authors.initials: J
authors.lastname: Chen
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Maltby
authors.forename: Louise A
authors.initials: LA
authors.lastname: Kelly
authors.forename: Hartmut
authors.initials: H
authors.lastname: Weiler
authors.forename: Ralf
authors.initials: R
authors.lastname: Krahe
authors.forename: Linda K
authors.initials: LK
authors.lastname: Ashworth
authors.forename: Emilio
authors.initials: E
authors.lastname: Garcia
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D051379:Mice; D008822:Mice, Transgenic; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics*; D009130:Muscle, Smooth / Q000737:chemistry*; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases; D017422:Sequence Analysis, DNA; D000096985:Calponins
chemical_list: D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D012260:Ribonucleases; D000096985:Calponins