Gene Targeting Models To Study Ec Coupling

在钙诱导的钙释放过程中最重要的参与者是“心脏”ryanodine受体RyR2。这是一个大的蛋白质（500 kD），形成一个大小为30x30x15nm的四聚体通道。跨膜通道被认为是由c端形成的。使用标准方法，RyR2缺失小鼠产生胚胎致死表型10倍。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耦合机制无关。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、组织受限的零背景。为了避免胚胎致死性，我们制备了几种转基因系，这些转基因系将允许有条件的和可诱导的基因靶向，限制在特定的心脏谱系（例如心室肌细胞），并在所需的发育阶段（特别是成人）诱导。实现这一目标的工具是Cre重组酶- LoxP重组系统和四环素反式激活剂系统。三个转基因小鼠系需要获得空间和时间控制的诱导敲除系统。一只小鼠携带一种受四环素敏感启动子控制的Cre重组酶转基因。第二只小鼠含有一个谱系特异性启动子（α肌球蛋白重链或NCX1驱动的四环素激活因子），因此可以在指定时间进行组织特异性敲除。第三只小鼠含有一个靶向构建体，其LoxP位点位于RyR2内源基因的外显子两侧。使用报告基因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，并且这种系统受到强力霉素/四环素的调节。现在将含有RyR2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对转基因进行调控敲除。他们的表型，无论是在个体心肌细胞还是在整个动物水平上，都将被检查，以确定RyR2蛋白在EC偶联中的重要作用。最终，通过嵌合cDNA构建靶向固定序列，使RyR2的内源转录机制转录突变形式的蛋白质，可以实现对零表型的拯救。该系统的证据已经在没有RyR2基因的胚胎干细胞中产生，我们希望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RyR2蛋白结构和功能相关的具体问题。

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 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.