Generation and characterization of CD40L-modified Mice

CD40L 修饰小鼠的生成和表征

• 批准号: 8580086
• 负责人: LORI Ruth COVEY
• 金额: $ 7.47万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-15 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8580086
• 关键词: 3' Untranslated Regions; Affect; Antibodies; Antibody Affinity; Antibody Formation; Antigen-Presenting Cells; Area; Autoimmune Process; B cell differentiation; B-Cell Neoplasm; B-Lymphocytes; Binding; Bone Marrow; CD4 Positive T Lymphocytes; CD40 Ligand; Cell Nucleus; Cells; Cellular Immunity; Complex; Cytoplasm; Data; Disease; Elements; Generations; Genes; Goals; Helper-Inducer T-Lymphocyte; Human; Humoral Immunities; Immune response; Indium; Inflammatory; Inflammatory Response; Lead; Link; Lymphocyte; Lymphoid; Lymphoid Tissue; Mediating; Messenger RNA; Mouse Strains; Mus; Mutant Strains Mice; Mutation; Organ; Pathogenesis; Pathway interactions; Peptide/MHC Complex; Play; Polypyrimidine Tract-Binding Protein; Population; Positioning Attribute; Process; Production; Publishing; RNA; RNA Binding; RNA Stability; Reagent; Regulation; Reporting; Research; Role; Signal Transduction; Spleen; Staging; Structure of germinal center of lymph node; T-Cell Activation; T-Lymphocyte; TNFRSF5 gene; TNFSF5 gene; Testing; Time; Transcript; Vaccines; base; blastocyst; defined contribution; design; embryonic stem cell; experience; in vivo Model; interest; lymph nodes; mRNA Decay; mRNA Stability; macrophage; malignant phenotype; mouse model; novel; novel strategies; pathogen; public health relevance; research study; response; screening

DESCRIPTION (provided by applicant): Historically, there has been a widespread appreciation of the functional significance of interactions between CD40 ligand (CD40L, CD154) on activated CD4+ T cells and CD40 expressed on antigen presenting cells (APC) in both humoral and cell-mediated immunity. However, more recent discoveries have underscored the significance of CD40-CD40L interactions in inflammatory responses and autoimmune pathogenesis. The fact that CD40L is central to both appropriate and inappropriate immune responses underscores the importance of having a comprehensive understanding of pathways that regulate CD40L expression and how different levels of CD40 engagement affect immune responses. The overall goal of this proposal is to develop a mouse model of variegated CD40L expression by replacing the endogenous CD40L gene with a gene lacking the RNA stability element in the 3' untranslated region (UTR) of the CD40L transcript (CD40L-¿5). Once the mouse is generated, experiments will test the effect of the CD40L-¿5 mutation on the humoral immune response. In particular, B cell populations in the spleen, lymph node and bone marrow will be analyzed and compared to these cell populations in littermate controls. The expression of CD40L in the mutant mouse will also be analyzed in the T follicular helper T (Tfh) cells since these cells are the critical subset of T helper cells required for the germinal center (GC) response. Key findings from our lab that are pertinent to the current proposal include, 1) the identification of an activation-induced pathway of CD40L mRNA decay; 2) the demonstration that this pathway is mediated by a PTB-containing complex (Complex I) binding to the CD40L mRNA at late times of activation; 3) the characterization of the stability element in both human and mouse CD40L transcripts; and 4) the finding that PTB is required for appropriate distribution of CD40L RNA between the nucleus and cytoplasm from both activated and na¿ve T cells. The proposed studies extend these preliminary and published findings by characterizing the PTB pathway of CD40L mRNA stability in an in vivo model. Results from these experiments will lead to a clearer understanding of factors controlling the expression of CD40L in humoral immunity and will uncover novel approaches for treating pathogen-related and autoimmune inflammatory diseases.

描述（由申请人提供）：从历史上看，在体液和细胞介导的免疫中，活化的CD 4 + T细胞上的CD 40配体（CD 40 L，CD 154）与抗原呈递细胞（APC）上表达的CD 40之间相互作用的功能意义已得到广泛认可。然而，最近的发现强调了CD 40-CD 40 L相互作用在炎症反应和自身免疫发病机制中的重要性。CD 40 L是适当和不适当免疫应答的核心，这一事实强调了全面了解调节CD 40 L表达的途径以及不同水平的CD 40参与如何影响免疫应答的重要性。本提案的总体目标是通过用在CD 40 L转录物的3'非翻译区（UTR）中缺乏RNA稳定性元件的基因（CD 40 L-5）替换内源性CD 40 L基因来开发多样化CD 40 L表达的小鼠模型。一旦产生小鼠，实验将测试CD 40 L-5突变对体液免疫应答的影响。特别是，将分析脾脏、淋巴结和骨髓中的B细胞群，并与同窝对照中的这些细胞群进行比较。还将在T滤泡辅助T（Tfh）细胞中分析突变小鼠中CD 40 L的表达，因为这些细胞是生发中心（GC）应答所需的T辅助细胞的关键亚群。我们实验室的主要发现包括：1）鉴定了活化诱导的CD 40 L mRNA衰减途径; 2）证明该途径由含PTB的复合物介导（复合物I）在活化后期与CD 40 L mRNA结合; 3）人和小鼠CD 40 L转录物中稳定元件的表征;和4）发现PTB是活化和幼稚T细胞的细胞核和细胞质之间CD 40 L RNA适当分布所必需的。拟议的研究通过表征体内模型中CD 40 L mRNA稳定性的PTB途径来扩展这些初步和已发表的发现。这些实验的结果将使我们更清楚地了解体液免疫中控制CD 40 L表达的因素，并将揭示治疗病原体相关和自身免疫性炎症性疾病的新方法。