Development Of Mouse Gene-targeting Models To Study Ec C

钙诱导的钙释放过程中的一个重要参与者是“心脏”兰尼碱受体RyR 2。这是一种大蛋白（500 kD），形成大小为30 × 30 × 15 nm的四聚体通道。跨膜通道被认为是由C-末端形成的。使用标准方法，我们已经创建并检查了缺乏RyR 2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。RyR 2敲除小鼠在10 dpc时产生胚胎致死表型。功能性研究正在进行中，以确定致死性的原因，因为它似乎与“经典”EC偶联机制无关。我们还建立了一个三方小鼠模型系统，以研究在体内的功能，该通道使用转基因的方法，以产生一个可诱导的，组织限制的空背景，在成年小鼠的心脏。为了避免胚胎致死性，我们制备了几种转基因品系，其将允许条件性和诱导性基因靶向，限于特定的心脏谱系（例如心室肌细胞）并且在期望的发育阶段（特别是在成人中）是可诱导的。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剂系统。已将含有RyR 2靶向载体的突变小鼠与调节性转基因小鼠杂交，以允许在成年小鼠中调节性敲除转基因;然而，动物显示不完全的转基因。他们的表型，无论是个别心肌细胞和整个动物的水平，将被检查，以确定EC耦合RyR 2蛋白的重要作用。最终，将通过用嵌合cDNA构建体靶向floxed序列以允许RyR 2的内源性转录机制转录蛋白质的突变形式来实现无效表型的拯救。该系统的证据已经在无RyR 2基因的ES细胞中产生，我们将利用该系统产生具有ryanodine受体突变形式的转基因小鼠，以研究与RyR 2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.