Abstract
The intergenic region between the mouse alpha-cardiac myosin heavy chain and beta-myosin heavy chain genes has previously been shown to direct expression of the bacterial chloramphenicol acetyltransferase reporter gene in transgenic mice in a tissue-specific manner. Sequence analyses located a putative myocyte-specific enhancer-binding factor (MEF-2) site situated in the regulatory region of this gene proximal to the start site of transcription. The role of this element in directing the cardiac compartment-specific expression of the transgene was assessed. The polymerase chain reaction was used to perform substitution mutagenesis of the MEF-2 binding site, and lack of MEF-2 binding was confirmed by gel retardation assays. The resultant construct was used to generate transgenic mice. Surprisingly, transgene expression was not down-regulated, but was significantly increased in the hearts of the MEF-2 mutant mice. In addition, cardiac-specific expression of the transgene was perturbed with significant levels of ectopic expression occurring in the aorta.
Highlights
Site-directed Mutagenesis of the MEF-2 Binding Site-Studies were initiated with the construct a-5.5, which includes -100 bp of the last intron of the P-cardiac myosin heavy chains (MHC) gene, the entire lastexon of the gene, the intergenic region, and theexons and introns thatcomprise the 5”untranslatedregion of the a-MHCgene, fused to thechloramphenicol acetyltransferase reporter gene
Synthesis of the MEF-2 Mutant Construct-These studies began with the constructa-5.5 described earlier, inwhich the entire intergenic region between the p- and a-MHCgenes was used to drive expression of cat [12]
The mutated MEF-2 sequence is shown in Fig. 1
Summary
Site-directed Mutagenesis of the MEF-2 Binding Site-Studies were initiated with the construct a-5.5 (clone 13 in Ref. 12), which includes -100 bp of the last intron of the P-cardiac MHC gene, the entire lastexon of the gene, the intergenic region, and theexons and introns thatcomprise the 5”untranslatedregion of the a-MHCgene, fused to thechloramphenicol acetyltransferase (cat) reporter gene. A 561-bp EcoRI-NdeI fragment present on the a-5.5 construct (-601 to -40 from the transcriptional start site), which contains the MEF-2 binding site, was subcloned into pSL301 (Invitrogen Corp., San Diego, CA), to make the construct pSLE561N. Site-directed mutagenesis of the MEF-2 binding site by overlap extension using the polymerase chain reaction (PCR) was performed as described [34]. PrimersMEF2-B and MEF2-C lay on the 561-bp EcoRI-NdeI fragment (-289 to -265 and -277 to -301, respectively) such that 13 bp were complementary to each other and contained the mutatedMEF-2 binding site. The sequences of these four primersare as follows, with the mutated MEF-2 site in underlined boldface type. At least three experiments were performed in each case; the values obtained did not vary by more than 20% from their respective means
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