In vivo analysis of the murine beta-myosin heavy chain gene promoter.

J Robbins,H Rindt,J Gulick,J Neumann,S Knotts

doi:10.1016/s0021-9258(18)53537-7

J Robbins, H Rindt + Show 3 more

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https://doi.org/10.1016/s0021-9258(18)53537-7

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Abstract

The 5' upstream region of the murine beta-myosin heavy chain (MHC) gene has been isolated and tested for its ability to drive gene expression in transgenic mice. Three classes of transgenic mice were generated. The constructs contained approximately 5000, 2500, and 600 base pairs of beta-MHC upstream sequence fused to the chloramphenicol acetyltransferase gene and were termed beta 5, beta 2.5, and beta .6, respectively. Muscle-specific expression was observed with all three constructs. However, only the beta 5 lines directed high levels of muscle-specific transgene expression in both pre- and postbirth mice. Expression driven by the two shorter constructs was two to three orders of magnitude lower when the chloramphenicol acetyltransferase specific activities were compared. These data suggest that a distal-positive element directs high levels of gene expression in the ventricle and in slow skeletal muscles. Analyses of transgene expression during heart maturation revealed that some of the beta 5 lines were not able to respond in an appropriate manner to developmental transcriptional cues. Unlike the endogenous beta-MHC gene, which is down regulated in the ventricles around the time of birth, reporter gene expression in the majority of the lines generated was not shut off in the ventricles of the adult animals. These data indicate that high levels of muscle-specific beta-MHC gene expression are dependent upon the combinatorial interactions of a number of sequence elements that are distributed over a large region of the gene's upstream sequence.

Highlights

From the $Department of Pharmacologyand Cell Biophysicsand the $Department of Molecular Genetics, Biochemistry,and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0575
3’, with the P-MHC gene preceding the a-MHC gene; the two heavy chain (MHC) gene has been isolated and tested genes are separated by about 4.5 kb of DNA [11,12,13]
Analysis of Transgenic Founder Animals-The offspring (Fo generation) from the implanted foster mice were screened by PCR as described previously [13]

Summary

Introduction

From the $Department of Pharmacologyand Cell Biophysicsand the $Department of Molecular Genetics, Biochemistry,and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0575. In all Transgene expression in the lines made with the shortest four p5 lines, under the assay conditions shown, CAT activity construct, containing only 600 bp of upstream DNA, varied was present in the heart, but noint the liver or in the lower significantly from line to line at thisdevelopmental stage, but hindleg which contains thesoleus muscle (Fig. 4A).We were, none of the four lines tested drove expression at the levels able to detect low levels of CAT activity in extracts observed for the p5 mice.

Results

Conclusion