Development of Mouse Gene-Targeting Models to Study EC Coupling

钙诱导中最重要的因素 钙释放过程是“心脏”兰尼定受体，RyR2。 这是一种形成四聚体通道的大蛋白(500kD) 尺寸为30x30x15 nm。跨膜通道被认为是 由C-末端形成。分子的大部分形成了大分子 跨越SR和SR之间的二分裂隙的“足突” 肌膜；其功能尚不清楚。我们计划研究一下 该通道的体内功能通过产生空的策略 背景通过(组织限制性的和可诱导的)敲除 野生型基因，紧随其后的是转基因拯救 组件。心脏的全球操作 激励-收缩耦合机制(例如，开发一种 可表达突变的RyR2的RyR2缺失的肌细胞) 产生一种胚胎致死的表型。其目标是诱导一种 小鼠心脏RyR2基因敲除的时空依赖性 这可以通过人类RyR2基因的突变形式来挽救。 为了避免胚胎死亡，我们采取了以下方法： 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)并可在所需的条件下诱导 发育阶段(尤其是成人)。这些工具可用于 实现这一点的是Cre重组酶-loxP重组 系统和四环素反式激活系统。一只老鼠是 构建了携带Cre重组酶转基因的 四环素敏感启动子和基因敲除的控制 含有loxP位点的构建体，其诱导 停用四环素后Cre的表达导致A基因的切除 目的基因的关键外显子。这个系统已经被控制住了 谱系特异性启动子(例如，心室肌球蛋白光 链MLC2V)，这样就可以进行组织特异性敲除 发生在指定的时间。目前有多个方正系列 含有四个Cre重组酶的构建 已确认身份。目前正在对这些线路进行适当的研究 表情。已经制备了MLC2V-TTA的构建载体，并 注射到合适的卵母细胞中。就RyR2而言 敲除后，小鼠129/SVJ基因组DNA片段已被 克隆、测序并证实含有RyR2的4个外显子。这个 突变小鼠RyR2靶向载体的制备 RyR2外显子包含两个侧翼loxP位点和一个pgk-neo 耐药阳性选择盒和pgk-tk负片 选择盒式磁带。RyR2基因打靶构建体已被 导入胚胎干细胞建立可诱导的RyR2 功能通道基因敲除小鼠体内功能的研究 心室肌中的兰尼定受体。这些细胞是 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.