Establishing and characterizing BAFF RFP reporter and BAFF knockin mice

描述（由申请人提供）：BAFF（B细胞活化因子，BLyS，TNFSF 13 B）是一种TNF家族细胞因子，由多种细胞类型如树突状细胞（DC）、单核细胞、嗜中性粒细胞、T和B细胞以及基质细胞产生。BAFF是成熟B细胞发育和对抗原（Ag）应答所必需的。BAFF表达的失调与许多免疫疾病的发展有关，最显著的是自身免疫性疾病，如系统性红斑狼疮（SLE）。正在开发和测试用于治疗与BAFF失调相关的疾病的新药。然而，关于何时、何地和如何在体内产生BAFF以及关于哪种产生BAFF的细胞有助于B细胞应答和B细胞相关疾病，相对知之甚少。我们将开发两种工具来监测BAFF的组织和细胞特异性表达，并评估哪些BAFF产生细胞有助于免疫应答和免疫病理学。我们有两个目标：目标1。BAFF-RFP报告基因和BAFF/BLyS敲入（KI）小鼠的产生。使用双重组酶载体，我们将首先产生一个转基因（Tg）线表达明亮，高度稳定的单体形式的红色荧光蛋白（RFP）命名为TagRFP-T的一个等位基因的Baff（Tnfsf 13 b）基因座。我们还将产生BAFF KI系（BAFF fl/+小鼠）并将这些小鼠与适当的B6-Cre系杂交以产生在DC或B细胞中缺乏BAFF表达的敲除（KO）小鼠。目标2.确定来源于DC或B细胞的BAFF在B细胞发育和体液免疫应答中的作用。我们将比较用T细胞依赖性Ag或T细胞非依赖性Ag免疫的WT小鼠、BAFF DC KO小鼠和BAFF B细胞KO小鼠的抗体应答。我们还将使用BAFF-RFP报告小鼠在免疫后随时间监测BAFF在不同细胞类型中的表达。由于BAFF-RFP报告基因和BAFFfl/fl KI小鼠将极大地增强基础B细胞生物学研究，并使临床前研究能够进行，以鉴定BAFF的功能重要来源以及治疗对BAFF产生和功能的影响，因此所提出的研究具有高度重要性，并具有产生高度影响的潜力。 公共卫生相关性：我们建议开发新的工具来测量可溶性因子BAFF的表达和功能，BAFF是B淋巴细胞存活和成熟所必需的。重要的是，B细胞产生重要的抗体，保护我们免受感染性疾病，也可能导致自身免疫性疾病，如类风湿性关节炎。这项工作可能有助于开发更好的疫苗和B细胞相关疾病的新疗法，如非霍奇金淋巴瘤，慢性淋巴细胞白血病，RA和系统性狼疮性肾炎。

DESCRIPTION (provided by applicant): BAFF (B cell activating factor, BLyS, TNFSF13B) is a TNF-family cytokine produced by a number of cell types such as dendritic cells (DCs), monocytes, neutrophils, T and B cells and stromal cells. BAFF is essential for mature B cells to develop and to respond to antigens (Ags). Dysregulation of BAFF expression has been implicated in the development of a number of immunologic diseases, most notably autoimmune diseases like systemic lupus erythematosus (SLE). New drugs are being developed and tested for treating diseases associated with BAFF dysregulation. However, relatively little is known about when, where and how BAFF is produced in vivo and about which BAFF-producing cells contribute to B cell responses and B cell associated diseases. We will develop two tools for monitoring tissue and cell specific expression of BAFF and for assessing which BAFF-producing cells contribute to immune responses and immunopathology. We have two Aims: Aim 1. Generation of BAFF-RFP reporter and BAFF/BLyS knockin (KI) mice. Using a dual recombinase vector we will first generate a transgenic (Tg) line expressing a bright, highly stable monomeric form of red fluorescent protein (RFP) designated TagRFP-T on one allele of the Baff (Tnfsf13b) locus. We will also create a BAFF KI line (BAFFfl/+ mice) and cross these mice with appropriate B6-Cre lines to create knockout (KO) mice lacking BAFF expression in either DCs or B cells. Aim 2. Determining the role of BAFF derived from DCs or B cells in B cell development and humoral immune responses. We will compare the antibody responses of WT mice, BAFF DC KO mice and BAFF B cell KO mice immunized with either a T cell-dependent Ag or a T cell-independent Ag. We will also monitor the expression of BAFF in different cell types over time after immunization using BAFF-RFP reporter mice. The proposed studies are highly significant and have the potential to have high impact since the BAFF-RFP reporter and BAFFfl/fl KI mice will greatly enhance basic B cell biology research and enable pre-clinical studies to be carried out to identify functionally important sources of BAFF and the effect of therapies on BAFF production and function. PUBLIC HEALTH RELEVANCE: We propose to develop new tools for measuring the expression and function of a soluble factor, BAFF, which is essential for the survival and maturation of B lymphocytes. Importantly, B cells produce vital antibodies that protect us against infectious diseases and also can contribute to autoimmune diseases like rheumatoid arthritis. This work may help in the development of better vaccines and new therapies for B cell-associated diseases such as non-Hodgkin's lymphoma, chronic lymphocytic leukemia, RA and systemic lupus erythematosis.