Mouse Genes that Regulate Hemostasis and/or Thrombosis

调节止血和/或血栓形成的小鼠基因

• 批准号: 7848325
• 负责人: THOMAS J. KUNICKI
• 金额: $ 29.65万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2010-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7848325
• 关键词: Adhesions; Animals; Back; Biological Assay; Bleeding time procedure; Blood; Blood Platelets; Blood Vessels; Blood flow; Candidate Disease Gene; Cardiovascular Diseases; Carotid Artery Injuries; Cells; Chromosomes, Human, Pair 4; Cloning; Coagulation Process; Collaborations; Collagen; Coronary Arteriosclerosis; DNA; DNA Resequencing; Data; Databases; Defect; Disease; Endothelial Cells; Exhibits; Failure; Fibroblasts; Foundations; GKLF protein; Gene Expression; Gene-Modified; Genes; Genetic; Genetic Polymorphism; Genetic Recombination; Genomics; Glycoproteins; Hematology; Hemorrhage; Hemostatic function; Human; In Vitro; Incidence; Individual; Institutes; Intention; Life; Location; Maps; Measures; Methods; Modeling; Molecular; Mus; Pattern; Penetrance; Phenotype; Platelet Glycoproteins; Platelet aggregation; Prevention; Proteins; RNA; Regulation; Research; Resolution; Resources; Risk; Severities; Smooth Muscle Myocytes; Stroke; System; Tail; Thrombosis; Thrombus; Time; Transgenic Mice; Whole Blood; Work; base; cell type; design; disease phenotype; experience; gene discovery; in vivo; in vivo Model; man; men; prevent; prototype; public health relevance; receptor; response; trait; transcription factor

DESCRIPTION (provided by applicant): Platelet GPVI is a pivotal receptor that initiates adhesion to collagen and propagates thrombus formation in vivo in both mice and men. A significant variability in GPVI expression and/or activity has been noted in both species. Elimination of the mouse gene Gp6 causes a uniform and expected defect in platelet responses to collagen in vitro, but on a mixed background, the in vivo phenotype is dichotomous, with either normal thrombus formation or severely impaired hemostasis. Using these "sensitized" mice, I have identified a single dominant locus, Modifier of hemostasis (MH), which is in linkage with the bleeding phenotype and maps to an 8 Mb region of chromosome 4. I propose that one or more modifier genes in MH regulate the in vivo phenotypes. To identify the modifier genes in this locus, I propose in Specific Aim 1.1, to positionally clone modifier gene(s) contributing to the in vivo phenotypes, through recombination cloning. Having obtained preliminary data that one of the candidate genes may be Klf4, the gene for Kruppel-like Factor 4, I have designed a strategy to evaluate in vivo the relevance of endothelial expression of Klf4 and any additional candidate genes that are identified. For this reason, Specific Aim 1.2 is to develop transgenic mice in which the relevance of the candidate modifier gene(s) can be assessed in vivo. These in vivo models will enable inducible (tetON), conditional (endothelial cell) knockdown or over expression of Klf4, as the prototype target. The models are adaptable to other relevant vascular cell types, such as smooth muscle cells or fibroblasts. In Specific Aim 2, I propose to use these transgenic mice to assess the relevance of the modifier gene products using in vivo thrombosis models. Emphasis will be placed on the results of the carotid artery injury model, with which I have the most experience, but alternative models can also be employed. The characterization of MH and the composite genes responsible for the bleeding/thrombotic phenotype will make a significant contribution to our understanding of the in vivo regulation of hemostasis. GPVI and other platelet receptors are implicated in many cases of human thrombosis. However, the severity of the disease phenotype is variable, suggesting the existence of modifying gene traits. Identification of genes that can modify the incidence and severity of thrombotic disease is of significant value to general hematology and cardiovascular disease. PUBLIC HEALTH RELEVANCE: Blood platelets are the smallest cellular component of the blood and are responsible for the formation of clots in humans and most animals, including mice. When working on mice for research purposes, we have the ability to add or remove genes and to thus find out how these genes influence the ability of platelets to form clots. This information is potentially valuable to our understanding of risk for and prevention of coronary artery disease and stroke in humans. I will take advantage of the mouse to remove one of the key genes that we believe controls platelet stickiness, and then I will add back into the same mice either the same human gene or a modified mouse gene. Then, when the appropriate system is in place, I can chemically induce an increase or a decrease in either the mouse or the human gene in a specific cell in the whole animal and measure the effect that this gene manipulation has on the formation of clots in that live mouse. The severity of coronary artery disease and stroke in man is variable, suggesting that there are differences between individuals in these modifying genes. It is my intention to discover gene differences in the mouse that are shared by humans and will help us predict the risk for or even prevent coronary artery disease or stroke in man.

描述（由申请人提供）：血小板GPVI是一种关键受体，在小鼠和人体内均可启动与胶原蛋白的粘附并促进血栓形成。GPVI表达和/或活性的显著变化在两个物种中均已观察到。小鼠基因Gp 6的消除在体外引起血小板对胶原蛋白的反应中的均匀和预期缺陷，但在混合背景下，体内表型是二分的，具有正常血栓形成或严重受损的止血。使用这些“致敏”小鼠，我已经确定了一个单一的显性基因座，止血修饰（MH），这是在连锁出血表型和映射到染色体4的8 Mb区域。我建议，一个或多个修饰基因在MH调节体内表型。为了鉴定该基因座中的修饰基因，我在具体目标1.1中提出，通过重组克隆定位克隆对体内表型有贡献的修饰基因。已经获得的初步数据，候选基因之一可能是Klf 4，Kruppel样因子4的基因，我已经设计了一种策略，以评估在体内的相关性Klf 4和任何其他候选基因的内皮表达被确定。出于这个原因，具体目标1.2是开发转基因小鼠，其中候选修饰基因的相关性可以在体内进行评估。这些体内模型将使Klf 4的诱导型（tetON）、条件性（内皮细胞）敲低或过表达成为原型靶标。该模型适用于其他相关的血管细胞类型，如平滑肌细胞或成纤维细胞。在具体目标2中，我建议使用这些转基因小鼠来评估修饰基因产物与体内血栓形成模型的相关性。重点将放在颈动脉损伤模型的结果上，我对颈动脉损伤模型最有经验，但也可以采用替代模型。MH的表征和复合基因负责出血/血栓形成表型将作出重大贡献，我们的理解在体内调节止血。GPVI和其他血小板受体与许多人类血栓形成病例有关。然而，疾病表型的严重程度是可变的，这表明存在修饰基因性状。鉴定能够改变血栓性疾病的发生率和严重程度的基因对一般血液学和心血管疾病具有重要价值。公共卫生相关性：血小板是血液中最小的细胞成分，负责人类和大多数动物（包括小鼠）的凝块形成。当以小鼠为研究对象时，我们有能力添加或删除基因，从而发现这些基因如何影响血小板形成血栓的能力。这些信息对我们了解人类冠状动脉疾病和中风的风险和预防具有潜在价值。我将利用小鼠的优势，去除我们认为控制血小板粘性的关键基因之一，然后我将同样的人类基因或修改过的小鼠基因重新添加到相同的小鼠中。然后，当适当的系统就位时，我可以化学诱导整个动物特定细胞中小鼠或人类基因的增加或减少，并测量这种基因操作对活小鼠中血栓形成的影响。人类冠状动脉疾病和中风的严重程度是可变的，这表明这些修饰基因在个体之间存在差异。我的目的是发现人类共有的小鼠基因差异，并帮助我们预测甚至预防人类冠状动脉疾病或中风的风险。