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

描述(申请人提供)：粒细胞-巨噬细胞集落刺激因子(GM-CSF)已研究了40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，但它最为人所知的是作为一种促炎症细胞因子在免疫和炎症中的作用。GM-CSF生物学已经在免疫学的各个分支中得到了研究，如自身免疫、癌症、传染病和疫苗开发。在免疫系统中，GM-CSF具有基本的和非多余的作用，使其成为一个有吸引力的治疗靶点。针对GM-CSF的几项临床试验正在进行中，正在考虑启动更多的试验。已经产生了GM-CSF及其受体敲除小鼠，并为免疫系统的功能提供了重要的见解。然而，报告GM-CSF表达的转基因小鼠并不存在，这项提议的特定目标1是产生这样的小鼠。我们设计了GM-CSF报告(Gr)小鼠的遗传修饰，通过与现有的转基因小鼠杂交，很容易使下一代GM-CSF报告/命运报告(Gr/fr)小鼠成为可能，这构成了特定的目标2。事实证明，使用即使在表达停止后仍能报告基因表达的Fate-Report小鼠，对于研究以其他方式无法充分解决的问题是一种有价值的方法。我们希望在转基因小鼠中产生的主要概念创新是存在两个报告基因，一个报告正在进行的GM-CSF表达，另一个报告GM-CSF已经表达。这使得能够容易地区分当前和过去的表达，而不是现在使用的命运报告鼠标行的情况。Gr/fr小鼠将有正常的GM-CSF表达，从而可以在生理环境下对其生物学进行研究。在具体目标3中，我们将解决一个相关的生物学问题，没有Gr/fr小鼠就无法解决这个问题，举例说明利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细胞并不产生GM-CSF。目前尚不清楚Th细胞亚群的GM-CSF表达是稳定的还是短暂的，也不知道哪些因素(A)决定了GM-CSF的Th细胞的表达或(B)改变了其目前的GM-CSF表达状态。我们将检验Th细胞亚群具有稳定的GM-CSF表达或不表达表型的假设。我们希望在这个项目完成后，我们将产生并全面描述Gr和Gr/fr小鼠品系。我们还将使用Gr/fr小鼠回答重要的生物学问题，这将展示它们推动进展的能力，否则是不可能的。

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.