Expression of human smooth muscle calponin in transgenic mice revealed with a bacterial artificial chromosome. Am J Physiol Heart Circ Physiol 2002 May;282(5):H1793-803
Date
04/18/2002Pubmed ID
11959645DOI
10.1152/ajpheart.00875.2001Scopus ID
2-s2.0-0036088084 (requires institutional sign-in at Scopus site) 12 CitationsAbstract
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.
Author List
Miano JM, Kitchen CM, Chen J, Maltby KM, Kelly LA, Weiler H, Krahe R, Ashworth LK, Garcia EAuthor
Hartmut Weiler PhD Associate Professor in the Physiology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsBlotting, Western
Calcium-Binding Proteins
Cells, Cultured
Chromosomes, Artificial, Bacterial
Gene Expression Regulation
Humans
Immunohistochemistry
In Situ Hybridization
Mice
Mice, Transgenic
Microfilament Proteins
Molecular Sequence Data
Muscle Proteins
Muscle, Smooth
Organ Specificity
Polymerase Chain Reaction
Restriction Mapping
Reverse Transcriptase Polymerase Chain Reaction
Ribonucleases
Sequence Analysis, DNA