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Am J Physiol Heart Circ Physiol 282: H1793-H1803, 2002. First published January 3, 2002; doi:10.1152/ajpheart.00875.2001
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Vol. 282, Issue 5, H1793-H1803, May 2002

Expression of human smooth muscle calponin in transgenic mice revealed with a bacterial artificial chromosome

Joseph M. Miano1, Chad M. Kitchen1, Jiyuan Chen1, Kathleen M. Maltby1, Louise A. Kelly2, Hartmut Weiler3, Ralf Krahe4, Linda K. Ashworth5, and Emilio Garcia5

1 Center for Cardiovascular Research, University of Rochester Medical Center, Rochester, New York 14642; 2 Department of Physiology, Medical College of Wisconsin, Milwaukee 53226; 3 Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53201-2178; 4 Division of Human Cancer Genetics, Ohio State University, Columbus, Ohio 43210, and 5 Human Genome Center, Lawrence Livermore National Laboratory, Livermore, California 94550

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.

promoter; development; genome


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