Adolph E A, Subramaniam A, Cserjesi P, Olson E N, Robbins J
Department of Medicine, University of Cincinnati College of Medicine, Ohio 45267-0575.
J Biol Chem. 1993 Mar 15;268(8):5349-52.
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.
小鼠α-心肌肌球蛋白重链和β-心肌肌球蛋白重链基因之间的基因间区域此前已被证明能够以组织特异性方式指导转基因小鼠中细菌氯霉素乙酰转移酶报告基因的表达。序列分析在该基因转录起始位点近端的调控区域定位到一个假定的心肌细胞特异性增强子结合因子（MEF-2）位点。评估了该元件在指导转基因心脏特异性表达中的作用。利用聚合酶链反应对MEF-2结合位点进行取代诱变，并通过凝胶阻滞试验证实缺乏MEF-2结合。将所得构建体用于产生转基因小鼠。令人惊讶的是，转基因表达并未下调，而是在MEF-2突变小鼠的心脏中显著增加。此外，转基因的心脏特异性表达受到干扰，在主动脉中出现了显著水平的异位表达。
