Development Of Mouse Gene-targeting Models To Study Ec C

钙诱导的钙释放过程中最重要的参与者是“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，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。三个转基因小鼠系是必要的，以获得空间和时间控制的诱导型敲除系统。一只小鼠携带在四环素敏感启动子控制下的Cre重组酶转基因。第二只小鼠含有谱系特异性启动子（α肌球蛋白重链或NCX 1驱动的四环素反式激活因子），因此可以在指定时间进行组织特异性敲除。第三只小鼠含有具有LoxP位点的靶向构建体，所述LoxP位点位于RyR 2内源基因的外显子的侧翼。使用报告基因ROSA 26小鼠，我们已经确定Cre蛋白酶的心脏限制性表达确实已经实现，并且这样的系统受到多西环素/四环素的调节。现在将含有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。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。

The most 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, 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. Three transgenic mouse lines were necessary to obtain spatial and temporal control of an inducible knock-out system. One mouse carries a Cre recombinase transgene under control of a tetracycline-sensitive promoter. The second mouse contains a lineage-specific promoter (alpha myosin heavy chain or NCX1 driven tetracycline tranactivator), so that a tissue-specific knockout can be made to occur at a specified time. The third mouse contains a targeting construct with LoxP sites flanking an exon of the RyR2 endogenous gene. Using the reporter ROSA26 mouse, we have determined that cardiac-restricted expression of Cre Recombinase has indeed been achieved and that such a system is regulated by doxycycline/tetracycline. The mutant mice containing the RyR2 targeting vector are now being crossed with the regulatory transgenic mice to permit regulated knock-out of the transgene in the adult mouse. 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.