Construction of analytical system for cardiac function of the phospholamban knock-out mouse

我们一直在研究磷磷的生理特性。为了了解它在完整心脏中的作用，我们尝试创建pln敲除小鼠。在第一步，我们必须研究PLN的转录调控，因为它在心肌细胞中的独特表达。我们分析了磷蛋白基因的5'上游区域。使用一系列的缺失结构，我们证明了-96到78个碱基对包含CCAAT基序的区域对该基因的转录至关重要。该区域与从大鼠心脏中提取的核蛋白特异性结合，使用竞争性寡核苷酸、抗体和重组蛋白进行凝胶转移分析表明，该区域与CCAAT基序结合蛋白中的NF-YA和NF-YB结合。在转染编码NF-YA和NF-YB的反义cdna的细胞中，这种区域依赖性转录减少到与转染相同意义cdna的细胞中所见的大约50%。因此，我们得出结论，跨越-96至-78碱基对的区域在磷蛋白基因的表达中起关键作用，该基因受核蛋白NF-Y的结合调节。第二步，我们利用第一步观察到的结果设计的PLN基因DNA构建体开始构建PLN敲除小鼠。

We have been studying the properties of phospholamban (PLN) in regard to its physiological aspects. To understand its role in the intact heart, we attempted to create the PLN-knock out mouse. In the first step, we had to study the transcriptional regulation of PLN because of its unique expression in the cardiac myocytes. We analyzed a 5'-upstream region of the phospholamban gene. Using a series of deletion constructs, we demonstrated that the region from -96 to-78 base pairs containing the CCAAT motif is essential for transcription of this gene. This region specifically binds to nuclear proteins extracted from rat hearts, and gel shift assays using competitive oligonucleotides, antibodies and recombinant proteins show that this region binds to the NF-YA and NF-YB among the CCAAT motif-binding proteins. This region-dependent transcription in cells transfected with antisense cDNAs encoding NF-YA and NF-YB was decreased to approximately 50% of that seen in cells transfected with the same sense cDNAs. We therefore conclude that the region spanning -96 to -78 base pairs plays a critical role in expression of the phospholamban gene which is regulated by binding of the nuclear protein NF-Y.In the second step, we started creating PLN-knock-out mouse by using the DNA construct of PLN gene designed by the result observed in the first step.