Development Of Mouse Gene-targeting Models To Study Ec C

在钙诱导的钙释放过程中，一个重要的角色是“心脏”兰尼定受体RyR2。这是一个大的蛋白质(500kD)，它形成一个大小为30x30x15 nm的四聚体通道。跨膜通道被认为是由C-末端形成的。使用标准的方法，我们已经创造并检测了缺乏RyR2的ES细胞，以表明这种蛋白质在发育过程中对心率的调节至关重要。RyR2基因缺失的小鼠在10DPC时产生胚胎致死表型。功能研究正在进行中，以确定致命性的原因，因为它似乎与‘经典的’EC偶联机制无关。我们还建立了一个三方小鼠模型系统来研究该通道的体内功能，使用转基因方法在成年小鼠心脏中产生可诱导的、组织受限的空背景。为了避免胚胎死亡，我们准备了几个转基因株系，它们将允许有条件的和可诱导的基因靶向，仅限于特定的心脏谱系(如心室肌细胞)，并可在所需的发育阶段(特别是成年)诱导。实现这一点的工具是Cre重组酶-loxP重组系统和四环素反式激活系统。含有RyR2靶向载体的突变小鼠已经与调控转基因小鼠杂交，以允许成年小鼠调控敲除转基因；然而，动物表现出不完全外显。他们的表型，包括单个心肌细胞和整个动物水平，都将被检测，以确定RyR2蛋白在EC偶联中的重要作用。最终，无效表型的挽救将通过靶向嵌合的cDNA结构的花序列来实现，以允许RyR2的内源转录机制转录突变形式的蛋白质。这个系统的证据已经在ES细胞中产生，RyR2基因是空的，我们将利用这个系统产生具有突变形式的ryanodine受体的转基因小鼠，以研究与RyR2蛋白的结构和功能相关的特定问题。

An important player in the calcium induced calcium release process is the "cardiac" ryanodine receptor, RyR2. This is a large protein (500 kD) which forms a tetrameric channel 30x30x15 nm in size. The trans-membrane channel is believed to be formed by the C-terminal. Using standard approaches, we have created and examined ES cells lacking RyR2 to show that this protein is critical in the regulation of heart rate during development. RyR2 null mice produce an embryonic-lethal phenotype by 10 dpc. Functional studies are on-going to determine the reason for the lethality, as it does not appear to be linked to a 'classic' EC coupling mechanism. We have also established a tripartite mouse model system to study the in-vivo function of this channel using a transgenic approach to produce an inducible, tissue-restricted null background by in adult mice hearts. To avoid embryonic lethality, we prepared several transgenic lines that would permit conditional and inducible gene targeting, limited to specific cardiac lineages (e.g. ventricular myocytes) and inducible at a desired developmental stage (particularly in the adult). The tools to accomplish this were the Cre recombinase - LoxP recombination system and the tetracycline trans-activator system. The mutant mice containing the RyR2 targeting vector have been crossed with the regulatory transgenic mice to permit regulated knock-out of the transgene in the adult mouse; however, the animals show incomplete penetrance. Their phenotype, both of individual cardiac myocytes and at a whole animal level, will be examined to determine the essential role of the RyR2 protein on EC coupling. Ultimately, rescue of the null-phenotype will be achieved through targeting of the floxed sequences with chimeric cDNA constructs to permit the endogenous transcriptional machinery of the RyR2 to transcribe mutant forms of the protein. Proof of this system has already been generated in ES cells null for the RyR2 gene, and we will exploit this system to generate transgenic mice with mutant forms of the ryanodine receptor to study specific questions related to the structure and function of the RyR2 protein.