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

我们一直在研究受磷蛋白（PLN）在其生理方面的特性。为了了解其在完整心脏中的作用，我们尝试创建PLN敲除小鼠。在第一步中，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。我们分析了受磷蛋白基因的5 '上游区域。使用一系列的缺失构建体，我们证明了从-96到-78碱基对的区域包含CCAAT基序是该基因转录所必需的。该区域特异性地结合从大鼠心脏提取的核蛋白，并且使用竞争性寡核苷酸、抗体和重组蛋白的凝胶迁移分析显示，该区域结合CCAAT基序结合蛋白中的NF-YA和NF-YB。在用编码NF-YA和NF-YB的反义cDNA转染的细胞中，这种区域依赖性转录降低至用相同正义cDNA转染的细胞中所见的约50%。因此，我们得出结论，跨越-96至-78碱基对的区域在受磷蛋白基因的表达中起着关键作用，受磷蛋白基因的表达受核蛋白NF-γ的结合的调节。在第二步中，我们开始通过使用由第一步中观察到的结果设计的PLN基因的DNA构建体来创建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.