Development of Mouse Gene-Targeting Models to Study EC Coupling

在钙诱导的钙释放过程中，最重要的角色是“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。使用标准方法，RyR2基因缺失的小鼠产生胚胎致死表型。因此，我们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。三个转基因小鼠品系是获得可诱导基因敲除系统的空间和时间控制所必需的。一只小鼠在四环素敏感启动子的控制下携带Cre重组酶转基因。第二只小鼠含有谱系特异性启动子(肌球蛋白重链或肌球蛋白轻链2v启动子驱动的四环素反式激活剂)，因此可以在特定时间进行组织特异性基因敲除。第三只小鼠包含一个靶向结构，其loxP位点位于RyR2内源基因的一个外显子两侧。利用报道的ROSA26小鼠，我们已经确定Cre重组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。含有RyR2靶向载体的突变小鼠现在正在与调节性转基因小鼠杂交，以允许成年小鼠中受调控的转基因敲除。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。最后，通过以嵌合的cDNA结构为靶点的花序列来挽救无效表型，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，在接下来的一年里，我们希望利用这个系统来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。

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. We have therefore 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 myosin light chain 2v promoter 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. Finally, 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 during the coming year, we hope to 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.