Visualization of CXCR3 allelic usage in vivo

CXCR3 等位基因体内使用的可视化

• 批准号: 7957584
• 负责人: Abhay R Satoskar
• 金额: $ 7.63万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957584
• 关键词: Alleles; Allografting; Animal Model; Animals; Autoimmune Diseases; Autoimmune Process; Binding; Bite; CD8B1 gene; CXC Chemokines; CXC chemokine receptor 3; CXCL10 gene; CXCL11 gene; CXCL9 gene; Cells; Communicable Diseases; Complex; Cutaneous; Data; Dendritic Cells; Disease; Elements; Ensure; Female; Flow Cytometry; Gender; Genes; Goals; Gonadal Steroid Hormones; Imagery; Immune; Immune response; Immunity; Immunology; Immunotherapeutic agent; Infection; Infection Control; Inflammation; Inflammatory; Inflammatory Response; Internal Ribosome Entry Site; Laboratories; Leishmania; Leishmania major; Leishmaniasis; Leukocyte Chemotaxis; Leukocytes; Life; Ligands; Link; Lupus; Maps; Mediating; Microscopy; Modeling; Mouse Strains; Multiple Sclerosis; Mus; Natural Killer Cells; Parasites; Pathogenesis; Predisposition; Process; Regulation; Regulatory T-Lymphocyte; Reporter; Research Personnel; Resistance; Rheumatoid Arthritis; Role; Sand Flies; Sex Characteristics; Site; T-Lymphocyte; Technology; Viral; Visceral Leishmaniasis; X Chromosome; X Inactivation; allograft rejection; cancer genetics; cell type; chemokine; chemokine receptor; enhanced green fluorescent protein; eosinophil; global health; in vivo; interest; male; neoplastic; novel; obligate intracellular parasite; pathogen; public health relevance; receptor; red fluorescent protein; response; sex; tool

DESCRIPTION (provided by applicant): The CXC chemokine receptor 3 (CXCR3) is expressed on: plasmacytoid dendritic cells (pDCs), eosinophils, NK cells, natural regulatory T cells, CD4+ and CD8+ T cells. The three main ligands of this receptor are the CXC chemokines CXCL9 (Mig), CXCL10 (IP-10) and CXCL11 (I-TAC), which are produced in high levels during inflammation. CXCR3 mediates immunity against pathogens such as Leishmania major by regulating the recruitment and function of effector T cells. However, CXCR3 also contributes to the pathogenesis of allograft rejection and many autoimmune diseases by promoting recruitment of pathogenic immune cells. The CXCR3 gene is mapped to chromosome X, and is therefore subject to monoallelic expression in females through random X chromosome inactivation (XCI). However, it is well documented that a number of X-linked genes "escape" X-chromosome inactivation and are expressed from both X chromosomes. If CXCR3 is amongst the genes which escape X-inactivation in immune cells, there will be quantitative differences in CXCR3 allele expression between the sexes. This could contribute to sex-associated differences in a variety of diseases. Gender differences in susceptibility to infectious and autoimmune diseases are well documented. Females are more resistant than males to infections caused by intracellular pathogens such as Leishmania, but they are more prone to autoimmune diseases such as rheumatoid arthritis. Our laboratory is studying the role of CXCR3 in different forms of leishmaniasis and is interested in determining its contribution to gender- associated resistance of females to this disease. We are interested in visualizing CXCR3 alleles that are used by different immune cells in females in vivo to determine whether the CXCR3 gene undergoes X-inactivation or escapes it. Our ability to study this complex process in vivo will be greatly enhanced if we are able to track CXCR3 expressing cells in mice and simultaneously visualize the alleles responsible for CXCR3 expression. The goals of this RO3 application are to generate a CXCR3-EFGP/RFP (CXCR3EGFP/RFP) dual reporter mouse carrying CXCR3 alleles which are linked to EGFP and RFP, respectively (Aim 1), and then to track the use of CXCR3 alleles in immune cells using an experimental L. major infection model (Aim 2). We have recently generated a CXCR3-bicistronic EGFP reporter (CIBER) mouse which expresses CXCR3 linked to enhanced green fluorescent protein (EGFP) via a viral IRES element. Our preliminary data show that CXCR3 expressing cells can be easily tracked in CIBER mice by flow cytometry and microscopy. In this project, we will generate a CXCR3-red fluorescent protein (RFP) reporter mouse and then cross it with a CIBER mouse to obtain a CXCR3EGFP/RFP mouse. This dual reporter mouse will carry one CXCR3 allele linked to EGFP and the other to RFP. Our preliminary data and the availability of CIBER mice demonstrate the feasibility of accomplishing the goals of this project within 2 years. CXCR3 EGFP/RFP reporter mice will allow us to visualize the differential expression of CXCR3 alleles in various immune cell types during infection. These mice will also be invaluable tools for other investigators to study the in vivo role of CXCR3 in infectious, inflammatory and neoplastic diseases. PUBLIC HEALTH RELEVANCE: The CXC chemokine receptor 3 is critical of leukocyte chemotaxis and inflammation associated with infection and autoimmune diseases. This project will develop a novel mouse strain which will allow visualization of CXCR3 expressing cells and study regulation of CXCR3 gene in living animal during inflammation and infection. This information will be important for developing immunotherapeutic approaches to treat inflammatory and infectious diseases.

描述（由申请人提供）：CXC趋化因子受体3（CXCR3）以：浆细胞样树突状细胞（PDC），嗜酸性粒细胞，NK细胞，天然调节T细胞，CD4+和CD8+ T细胞。该受体的三个主要配体是CXC趋化因子CXCL9（MIG），CXCL10（IP-10）和CXCL11（I-TAC），它们在炎症过程中以高水平产生。 CXCR3通过调节效应T细胞的募集和功能来介导对病原体（例如Leishmania Major）的免疫力。但是，CXCR3还通过促进致病性免疫细胞的募集来有助于同种异体移植排斥和许多自身免疫性疾病的发病机理。 CXCR3基因映射到染色体X，因此通过随机X染色体灭活（XCI）在女性中受到单位型表达。但是，有充分的文献证明，许多X连锁基因“逃脱” X染色体失活，并从两个X染色体中表达。如果CXCR3是逃避免疫细胞中X灭活的基因之一，则性别之间的CXCR3等位基因表达将存在定量差异。这可能导致各种疾病中的性别相关差异。有充分记录在感染性和自身免疫性疾病的敏感性方面的性别差异。女性比男性对由利什曼原虫等细胞内病原体引起的感染更具抵抗力，但它们更容易发生自身免疫性疾病，例如类风湿关节炎。我们的实验室正在研究CXCR3在不同形式的利什曼病中的作用，并有兴趣确定其对女性对这种疾病的性别抗药性的贡献。我们有兴趣可视化体内女性中不同免疫细胞使用的CXCR3等位基因，以确定CXCR3基因是否经历X灭活或逃脱。如果我们能够跟踪小鼠中表达细胞的CXCR3并同时可视化负责CXCR3表达的等位基因，那么我们在体内研究这一复杂过程的能力将大大增强。该RO3应用的目标是生成CXCR3-EFGP/RFP（CXCR3EGFP/RFP）双报告鼠标，分别载有CXCR3等位基因，这些鼠标分别链接到与EGFP和RFP相关的CXCR3等位基因（AIM 1）（AIM 1），然后使用CXCR3等位基因在免疫细胞中使用实验性l. Major l. Major aim aim aim aim aim aim aim（aim 1），然后跟踪使用CXCR3等位基因。我们最近通过病毒IRES元素生成了CXCR3-BICISTRONIC EGFP报告基因（Ciber）小鼠，该CXCR3与增强的绿色荧光蛋白（EGFP）相关。我们的初步数据表明，通过流式细胞仪和显微镜可以轻松地在Ciber小鼠中跟踪CXCR3表达细胞。在此项目中，我们将生成CXCR3-红色荧光蛋白（RFP）报告基因鼠标，然后用Ciber小鼠交叉以获得CXCR3EGFP/RFP小鼠。该双报告鼠标将携带一个与EGFP链接的CXCR3等位基因，另一个与RFP链接的等位基因。我们的初步数据和Ciber小鼠的可用性证明了在2年内实现该项目目标的可行性。 CXCR3 EGFP/RFP报告基因小鼠将使我们能够可视化感染过程中各种免疫细胞类型中CXCR3等位基因的差异表达。这些小鼠也将是其他研究人员研究CXCR3在感染，炎症和肿瘤疾病中的体内作用的宝贵工具。 公共卫生相关性：CXC趋化因子受体3批评白细胞趋化性和与感染和自身免疫性疾病有关的炎症。该项目将开发出一种新型的小鼠菌株，该菌株将允许在炎症和感染期间可视化CXCR3表达细胞并研究活动物中CXCR3基因的调节。这些信息对于开发免疫治疗方法来治疗炎症和传染病很重要。