Establishing and characterizing BAFF RFP reporter and BAFF knockin mice

描述(申请人提供)：BAFF(B细胞激活因子，BLyS，TNFSF13B)是一种由多种细胞类型产生的肿瘤坏死因子家族细胞因子，如树突状细胞(DC)、单核细胞、中性粒细胞、T和B细胞以及基质细胞。BAFF对于成熟B细胞的发育和对抗原(AGS)的反应是必不可少的。BAFF表达异常与许多免疫性疾病的发生有关，尤其是系统性红斑狼疮(SLE)等自身免疫性疾病。正在开发和测试治疗与BAFF调节失调相关的疾病的新药。然而，关于BAFF在体内何时、何地和如何产生，以及哪些产生BAFF的细胞对B细胞反应和B细胞相关疾病有贡献，人们知之甚少。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产生细胞对免疫反应和免疫病理学有贡献。目的1.制备BAFF-RFP报告基因和BAFF/BLyS敲击(KI)小鼠。使用双重组酶载体，我们将首先建立一个转基因(TG)系，在Baff(Tnfsf13b)基因座的一个等位基因上表达一种明亮、高度稳定的红色荧光蛋白(RFP)单体形式，命名为TagRFP-T。我们还将创建一个BAFF KI系(BAFFfl/+小鼠)，并将这些小鼠与适当的B6-Cre系杂交，以创建在DC或B细胞中缺乏BAFF表达的敲除(KO)小鼠。目的2.确定DC或B细胞来源的BAFF在B细胞发育和体液免疫反应中的作用。我们将比较WT小鼠、BAFF DC KO小鼠和BAFF B细胞KO小鼠对T细胞依赖抗原和T细胞非依赖抗原免疫的抗体反应。我们还将使用BAFF-RFP报告鼠在免疫后一段时间内监测不同类型细胞中BAFF的表达。由于BAFF-RFP报告小鼠和BAFFfl/fl Ki小鼠将极大地促进基础B细胞生物学研究，并使临床前研究得以开展，以确定BAFF的重要功能来源以及治疗对BAFF生产和功能的影响，因此拟议的研究具有非常重要的意义和潜在的影响。

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.