Regulation of Angiogenesis and Renin Expression in Rats

大鼠血管生成和肾素表达的调节

• 批准号: 7217710
• 负责人: ANDREW S. GREENE
• 金额: $ 43.81万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7217710
• 关键词: angiogenesis; angiotensin /renin /aldosterone hypertension; artificial chromosomes; bioinformatics; dietary sodium; enzyme activity; functional /structural genomics; gene mutation; genetic manipulation; genetic polymorphism; genetically modified animals; inbreeding; laboratory rat; nucleic acid sequence; nutrition related tag; tissue /cell culture; transfection

A myriad of physiological data confirms that renin is a critical component of the normal angiogenesis response seen in skeletal muscle with electrical stimulation. Using SS-13BN/Mcwi (SS-13BN) consomic rats and a series of congenic rats, we have demonstrated that restoration of a small region surrounding the renin gene confers normal renin levels and restores the normal angiogenic phenotype in the Dahl S (SS) rat. Despite this data, no difference has been observed in the coding region or the classically defined promoter region of the renin gene that would explain the differences in expression. Therefore it is the goal of this application to determine the sequence variants in the region, demonstrate which of these variants impact renin gene regulation in vitro, and using a transgenic approach, demonstrate that this candidate sequence variant eliminates normal renin regulation and the angiogenic phenotype in a subcongenic rat. This will be accomplished through a series of experiments comprising three specific aims. In specific Aim 1, we will begin with a congenic rat line SS.BN- (D13rat123-D13rat101)/Mcwi (referred to as line 9) in which a region of less than 4.5 Mbp surrounding the renin gene region has been introgressed from the BN (Brown Norway) genome onto the SS/JrHsdMcwi (SS) background. Using well-established techniques of marker-assisted selection to identify recombinants, we will reduce this candidate region to 1-2 Mbp. In specific Aim 2, we will identify candidate sequence variants within this reduced region by sequencing the SS and other closely related strains that do not share the antiangiogenic phenotype, including the Dahl salt resistant (SR), the Lyon Normotensive (LN), the Fawn Hooded Hypertensive (FHH) and the BN rats. Using the five strains, we have shown that we can reduce the number of potential causative strain-specific sequence variants within the candidate region to a manageable number, theoretically as low as 10, with a probability of having a false positive candidate mutation remaining below 1%. The candidate variants identified by sequencing and validated by a bioinformatics approach will be tested in vitro. This will be achieved by the use of a cell based system in which endothelial cells derived from the subcongenic line carrying the reduced region (Aim 1) will be transfected with SS Bacterial Artificial Chromosomes (BACs) and renin regulation will be assessed. We will use the surrogate phenotype of serum starvation-induced renin expression to assess the efficacy of the targeted mutation in modulating the renin phenotype. Specific Aim 3 will focus on the final identification and validation of the causative strain-specific sequence variant in this region. Our final experiment will be to use a transgenic rescue approach with a bacterial artificial chromosome transgene harboring the SS allele. Substitution of the allele identified in previous experiments will be performed on the background of the reduced subcongenic to test for confirmation of the loss of the angiogenic phenotype in vivo.

大量生理数据证实肾素是正常血管生成反应的关键组成部分 在电刺激的骨骼肌中可见。使用SS-13BN/Mcwi (SS-13BN) 体鼠等系列 在同系大鼠中，我们已经证明，肾素基因周围小区域的恢复赋予 Dahl S (SS) 大鼠中肾素水平正常并恢复正常血管生成表型。尽管有这些数据，但没有 在肾素基因的编码区或经典定义的启动子区中观察到差异 这可以解释表达上的差异。因此，本应用程序的目标是确定 该区域的序列变异，证明这些变异中哪些变异影响体外肾素基因调控，以及 使用转基因方法，证明该候选序列变体消除了正常肾素 亚同类大鼠的调节和血管生成表型。这将通过一系列的工作来实现 实验包括三个具体目标。在具体目标 1 中，我们将从同类大鼠系 SS.BN- 开始 (D13rat123-D13rat101)/Mcwi(称为第9行)，其中围绕 肾素基因区域已从 BN（挪威棕色）基因组渗入到 SS/JrHsdMcwi (SS) 上 背景。使用成熟的标记辅助选择技术来鉴定重组体，我们将 将此候选区域减少到 1-2 Mbp。在具体目标 2 中，我们将识别候选序列变体 通过对 SS 和其他不具有抗血管生成能力的密切相关菌株进行测序，可以发现这个缩小的区域 表型，包括达尔耐盐 (SR)、里昂正常血压 (LN)、小鹿连帽 高血压 (FHH) 和 BN 大鼠。使用这五种菌株，我们已经证明我们可以减少 将候选区域内潜在的致病菌株特异性序列变异控制到可管理的数量， 理论上低至 10，假阳性候选突变的概率保持在 1% 以下。 通过测序鉴定并通过生物信息学方法验证的候选变体将在体外进行测试。 这将通过使用基于细胞的系统来实现，其中内皮细胞源自 携带减少区域（目标 1）的亚同类系将用 SS 细菌人工转染 将评估染色体 (BAC) 和肾素调节。我们将使用血清的替代表型 饥饿诱导的肾素表达，以评估靶向突变调节肾素的功效 表型。具体目标 3 将重点关注致病菌株特异性的最终鉴定和验证 该区域的序列变异。我们的最终实验将是使用转基因救援方法 含有 SS 等位基因的细菌人工染色体转基因。中确定的等位基因的替换 之前的实验将在减少亚同源的背景下进行以进行验证 体内血管生成表型的丧失。