REGULATION OF SURROGATE LIGHT CHAIN IN AGED MICE

老年小鼠替代轻链的调节

• 批准号: 6626442
• 负责人: BONNIE B. BLOMBERG
• 金额: $ 30.3万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-15 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6626442
• 关键词: Abelson leukemia virus; B cell receptor; B lymphocyte; age difference; aging; animal old age; biological models; bone marrow; cell growth regulation; cell line; chromatin; gel mobility shift assay; genetic regulation; genetic regulatory element; genetic transcription; genetically modified animals; immunoglobulin genes; immunoglobulins; immunoprecipitation; laboratory mouse; mature animal; northern blottings; polymerase chain reaction; transcription factor; transfection; western blottings

DESCRIPTION (Applicant's Abstract): The antibody-mediated humoral immune response has been shown to be depressed in aged humans and experimental I animals creating increased morbidity and mortality in the aged population. The pre-B-cell receptor (pBCR), composed of the surrogate light chain (SLC, lambda.5 and VpreB) and the immunoglobulin (Ig) mu (mu) heavy (H) chain is critical for formation of pre-B-cells and for variable region gene (VH) selection. These studies will focus on the molecular mechanisms responsible for the decline in pre-B-cells, SLC and transcription factors seen in aged mice. We hypothesize that the decrease in pre-B-cells is a direct result of a lower amount of the SLC produced in the pro-B/early pre-B-cell stages, and that this is regulated at the transcription level. This proposal will compare pro-B/pre-B-cells from aged vs. young mice for the presence and function of transcription factors known to regulate lambda5 and VpreB, accessibility of lambda5 and VpreB chromatin, whether the defects are intrinsic to B lineage cells, and attempt to reverse the aged phenotype in transgenic mice. In Specific Aim I we will determine the regulation of transcription and transcription factors which affect SLC expression in senescent pro-B/pre-B-cells. Abelson Murine Leukemia Virus (A-MuLV) transformed pro-B/pre-B-cell lines will be prepared, and these, IL-7 expanded pro-B/pre-B-cells and ex vivo isolated (B220+ mu-) bone marrow cells from aged (>20 months) or young (2-4 months) mice will be used for this project. The mRNA stability and nascent transcripts of lambda5 and VpreB, the presence and function of transcription factors known (EBF and E47) and likely (Ikaros/Aiolos, Id) to be involved in lambda5/VpreB transcription regulation will be assayed by various methods including RT-PCR, electrophoretic mobility shift assay (EMSA), and transfection of lambda5/VpreB constructs. We will also measure the DNase I hypersensitivity and the histone acetylation status of the lambda5 and VpreB loci as a function of age. In Aim 2 we will assay possible stromal influences on the transcription regulation of lambda5/VpreB. Young and aged stromal preparations will be used to assess their influence on the levels of lambda5/VpreB transcription regulation in pro-B/pre-B-cells in vitro, as well as in vivo adoptive transfer studies of young and/or old B lineage precursors into young or old recipients. In Aim 3 we will prepare retroviral vectors and transgenic mice with lambda5/VpreB to attempt to rescue the aged B lineage phenotype. The experiments in this proposal will provide a molecular dissection of the abnormal regulation lambda5/VpreB which we hypothesize results in decreased pre-B-cells and an altered VH repertoire in aged mice.

描述（申请人的摘要）：抗体介导的体液免疫 已经证明反应在年龄的人类和实验I中受到抑郁 动物在老年人群中产生的发病率和死亡率增加。这 由替代光链组成的前B细胞受体（PBCR）（SLC， Lambda.5和VPREB）和免疫球蛋白（IG）MU（MU）重（H）链为 对于形成前B细胞和可变区域基因（VH）至关重要 选择。这些研究将集中于负责的分子机制 在老年小鼠中看到的B细胞，SLC和转录因子的下降。我们 假设B细胞的减少是较低的直接结果 在Pro-B/早期B细胞阶段产生的SLC数量，这是 在转录水平上进行调节。该建议将比较 来自老年小鼠的Pro-B/Pre-B细胞，用于 已知调节lambda5和vpreb的转录因子，可访问性 lambda5和vpreb染色质，缺陷是否固有到B谱系 细胞，并试图扭转转基因小鼠的老化表型。 在特定目的中，我将确定转录的调节和 影响SLC表达的转录因子 Pro-B/Pre-B细胞。 Abelson Murine白血病病毒（A-Mulv）转化了 将准备Pro-B/Pre-B细胞线，这些IL-7扩展了 Pro-B/Pre-B细胞和离体分离（B220+ MU-）骨髓细胞 （> 20个月）或年轻（2-4个月）将用于该项目。 mRNA lambda5和vpreb的稳定性和新生的转录本，存在和 已知转录因子的功能（EBF和E47），可能 （ikaros/aiolos，id）参与lambda5/vpreb转录法规 将通过包括RT-PCR，电泳迁移率在内的各种方法进行测定 移位测定（EMSA）和lambda5/vpreb构建体的转染。我们也会 测量DNase I超敏反应和组蛋白乙酰化状态 lambda5和vpreb基因座与年龄的关系。在AIM 2中，我们将可以测定 对lambda5/vpreb的转录调控的基质影响。年轻和 老化的基质制剂将用于评估其对水平的影响 lambda5/vpreb在体外pro-b/b-cell中的转录调节，AS 以及年轻和/或旧B血统的体内收养转移研究 前体进入年轻或老年接受者。在AIM 3中，我们将准备逆转录病毒 用lambda5/vpreb尝试营救老化的b，向量和转基因小鼠 谱系表型。该提案中的实验将提供分子 我们假设的异常调节lambda5/vpreb的解剖 导致BED前细胞减少和老年小鼠的VH曲目改变。