Expression of human smooth muscle calponin in transgenic mice revealed with a bacterial artificial chromosome.
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.
Miano
Joseph M
JM
Center for Cardiovascular Research, University of Rochester Medical Center, Rochester, New York 14642, USA. Joseph_Miano@urmc.rochester.edu
Kitchen
Chad M
CM
Chen
Jiyuan
J
Maltby
Kathleen M
KM
Kelly
Louise A
LA
Weiler
Hartmut
H
Krahe
Ralf
R
Ashworth
Linda K
LK
Garcia
Emilio
E
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
D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D012260:Ribonucleases; D000096985:Calponins