Development of GM-CSF reporter and reporter/fate-reporter mice

描述（由申请人提供）：粒细胞-巨噬细胞集落刺激因子（GM-CSF）已经被研究了四十多年，是研究最广泛的细胞因子之一。GM-CSF参与多种生理过程，从伤口愈合和造血到胎盘和胎儿发育，但最广为人知的是其在免疫和炎症中的促炎细胞因子作用。转基因csf生物学已在免疫学的各个分支中得到研究，如自身免疫、癌症、传染病和疫苗开发。在免疫系统中，GM-CSF具有重要和非冗余的作用，使其成为一个有吸引力的治疗靶点。一些针对GM-CSF的临床试验正在进行中，并且正在考虑启动更多的试验。GM-CSF及其受体敲除小鼠已经产生，并为免疫系统的功能提供了重要的见解。然而，报道表达GM-CSF的转基因小鼠尚不存在，本提案的Specific Aim 1就是产生这种小鼠。我们在GM-CSF报告基因（Gr）小鼠中设计了基因修饰，通过与现有转基因小鼠杂交，可以很容易地培育出下一代GM-CSF报告基因/命运报告基因（Gr/fr）小鼠，这构成了Specific Aim 2。使用命运报告小鼠，即使在表达停止后也报告基因表达，已被证明是研究无法充分解决的问题的一种有价值的方法。我们想要产生的转基因小鼠的主要概念创新是两个报告基因的存在，一个报告正在进行的GM-CSF表达，另一个报告GM-CSF已经表达。这可以很容易地区分当前和过去的表达，这与现在使用的命运报告鼠标系不同。Gr/fr小鼠将具有正常的GM-CSF表达，允许在生理背景下研究其生物学。在具体目标3中，我们将解决一个没有Gr/fr小鼠就无法解决的相关生物学问题，举例说明使用这些小鼠可以取得的进展。尽管GM-CSF的产生对Th细胞的功能至关重要，但大多数Th细胞不产生GM-CSF。目前尚不清楚Th细胞亚群在GM-CSF表达方面是否具有稳定或短暂的表型，也不知道哪些因素(a)决定GM-CSF的Th细胞表达或(b)改变其当前的GM-CSF表达状态。我们将检验Th细胞亚群具有稳定的表达gm - csf或-不表达表型的假设。我们期望在完成这个项目后，我们将生成并全面表征Gr和Gr/fr小鼠系。我们还将使用Gr/fr小鼠来回答重要的生物学问题，这将证明它们有能力促进其他方法无法实现的进步。

DESCRIPTION (provided by applicant): Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been studied for over forty years, and is among the most extensively researched cytokines. GM-CSF is involved in a variety of physiological processes ranging from wound healing and haematopoiesis to placental and fetal development, but it is best known for its role as a pro-inflammatory cytokine in immunity and inflammation. GM-CSF biology has been studied in various branches of immunology, such as autoimmunity, cancer, infectious diseases and vaccine development. In the immune system, GM-CSF has essential and non-redundant roles that make it an attractive therapeutic target. Several clinical trials targeting GM-CSF are underway, and initiation of additional trials is being considered. Both GM-CSF and its receptor knockout mice have been generated and have provided important insights into functioning of the immune system. However, transgenic mice that report GM-CSF expression do not exist, and Specific Aim 1 of this proposal is to generate such mice. We have designed genetic modifications in the GM-CSF reporter (Gr) mice that will readily enable subsequent generation of GM-CSF reporter/fate-reporter (Gr/fr) mice by crossing with existent transgenic mice, and this constitutes Specific Aim 2. The use of fate-reporter mice, which report gene expression even after expression ceases, has proved to be a valuable approach for studying questions that could not be adequately addressed otherwise. The principal conceptual innovation in the transgenic mice that we want to generate is the presence of two reporter genes, one that reports ongoing GM-CSF expression, the other reporting that GM-CSF has been expressed. This enables easy differentiation between current and past expression, which is not the case with fate-reporter mouse lines now being used. The Gr/fr mice will have normal GM-CSF expression, allowing studies of its biology in a physiological context. In Specific Aim 3 we will address a relevant biological question that cannot be addressed without Gr/fr mice, exemplifying advances that can be made by use of these mice. Even though GM-CSF production is crucial for function of Th cells, a majority of them do not produce GM-CSF. It is not known whether subpopulations of Th cells have stable or transient phenotype with regard to GM-CSF expression, nor is it known which factors (a) determine Th cell expression of GM-CSF or (b) change its current GM-CSF- expressing status. We will test the hypothesis that subpopulations of Th cells have stable GM-CSF-expressing or -non-expressing phenotype. We expect that upon completion of this project we will have generated and comprehensively characterized Gr and Gr/fr mouse lines. We will also use Gr/fr mice to answer important biological questions, which will demonstrate their capacity to facilitate advances that would not be feasible otherwise.