Development of Mouse Gene-Targeting Models to Study EC Coupling

在钙诱导的最重要的球员 钙释放过程是“心脏”ryanodine受体，RyR 2。 这是一个大蛋白（500 kD），形成一个四聚体通道 尺寸为30 x30 x15 nm。跨膜通道被认为是 由C-末端形成。大部分的分子形成了 “足突”，跨越SR和 肌膜;其功能未知。我们计划学习 该通道的体内功能是通过产生无效的策略来实现的 背景技术通过（组织限制性和诱导性）敲除 野生型基因，然后用基因改变的 件.心脏整体操作 兴奋-收缩耦合机制（例如，开发一个 其中可表达突变的RyR 2的RyR 2缺失肌细胞） 产生胚胎致死表型。目标是诱导一个 小鼠心脏RyR 2基因敲除时空依赖性 可以通过人类RyR 2 cDNA的突变形式来拯救。 为了避免胚胎死亡，我们正在采取以下方法： 条件性和诱导性基因靶向，仅限于特定心脏 谱系（例如心室肌细胞），并在所需的 发育阶段（尤其是成年阶段）。的工具 实现这一点的是Cre重组酶- LoxP重组 系统和四环素反式激活子系统。鼠标是 构建了一个Cre重组酶转基因载体， 四环素敏感启动子的控制以及敲除 构建体含有LoxP位点，以这样的方式， 四环素的撤回导致切除的Cre表达的a 目的基因的关键外显子。这个系统被置于控制之下 谱系特异性启动子（如心室肌球蛋白轻 链MLC 2 V），从而可以进行组织特异性敲除， 发生在特定的时间。目前已有多条创始人线路 含有tetop-Cre蛋白酶的构建体， 鉴定目前正在研究这些线路， 表情已经制备了MLC 2 V-tTA的构建体， 注射到合适的卵母细胞中。关于RyR 2 敲除后，15 kb小鼠129/SvJ基因组DNA片段已被 克隆、测序并确认含有RyR 2的4个外显子。的 已经制备了突变小鼠RyR 2靶向载体，其中 RyR 2外显子含有两个侧翼loxP位点和一个pGK-neo 耐药阳性选择盒以及pGK-tk阴性 选择卡匣。RyR 2基因靶向构建体已被 引入胚胎干细胞以建立诱导型RyR 2 功能性通道基因敲除小鼠，以研究 心室肌Ryanodine受体这些细胞 目前注射用于鉴定合适的嵌合体。

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. The bulk of the molecule forms the large "foot process" that spans the diadic cleft between the SR and sarcolemmal membranes; its function is unknown. We plan to study the in-vivo function of this channel by a strategy of producing a null background by (tissue restricted and inducible) knockout of the wild-type gene, followed by rescue with genetically altered components. Global manipulation of the cardiac excitation-contraction coupling machinery (e.g., developing an RyR2 null myocyte in which mutated RyR2 can be expresses) produces an embryonic-lethal phenotype. The goal is to induce a spatially and temporally dependent RyR2 knock-ou in mouse heart that can be rescued by mutated forms of the human RyR2 cDNA. To avoid embryonic lethality, we are taking the following approach: 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 are the Cre recombinase - LoxP recombination system and the tetracycline trans-activator system. A mouse is constructed which carries a Cre recombinase transgene under control of a tetracycline-sensitive promoter as well as a knockout construct containing LoxP sites in such a manner that induction of Cre expression by withdrawal of tetracycline causes excision of a critical exon of the target gene. This system is placed under control of a lineage-specific promoter (such as the ventricular myosin light chain MLC2V), so that a tissue-specific knockout can be made to occur at a specified time. Currently a number of founder lines containing a construct of tetop-Cre Recombinase have been identified. These lines are currently being studied for appropriate expression. A construct of MLC2V-tTA has been prepared and injected into the appropriate oocytes. In the case of the RyR2 knockout, a 15 kb mouse 129/SvJ genomic DNA fragment has been cloned, sequenced and confirmed to contain 4 exons of RyR2. The mutant mouse RyR2 targeting vector has been prepared in which RyR2 exons contain two flanking loxP sites and a pGK-neo resistant positive selection cassette as well as a pGK-tk negative selection cassette. The RyR2 gene targeting construct has been introduced into embryonic stem cells to establish inducible RyR2 functional channel knock-out mice to study the in vivo funciton of ryanodine receptor in ventricular myocardium. These cells are currently injected for identification of appropriate chimeras.