A novel and improved mouse system for studying macrophage specific gene deletion

描述（申请人提供）：巨噬细胞对组织稳态至关重要，在炎症、免疫和癌症中发挥重要作用，因此了解巨噬细胞生物学是理解稳态和疾病的基础。目前可用的靶向Cre表达的小鼠将flxed基因缺失到巨噬细胞中，主要基于驱动Cre表达的启动子，存在显著且严重的限制。1)目前在巨噬细胞中驱动Cre的所有可用启动子在整个髓系甚至其他细胞类型中都是活跃的，这往往比巨噬细胞中的启动子更强，因此缺乏巨噬细胞特异性；2)目前可用的启动子仅针对巨噬细胞的一个子集；3)由F4/80和LysM驱动的两种最常用的Cre系也被敲入一个内源性等位基因，导致表达弱，阻止纯合子小鼠的繁殖。本研究的目的是开发和表征一种新的改良转基因小鼠系，以特异性靶向巨噬细胞表达Cre。我们建议将人CD68启动子与巨噬细胞特异性增强子结合使用，以驱动活性改善的Cre和双电转录物中的dtTomato荧光蛋白的表达，用于体内跟踪和分选。转基因小鼠将广泛地具有报告基因和内源性floxed基因的特征，并与目前可用的表达cre的小鼠进行比较。因此，我们提出在C57BL/6菌株中开发一种新的真正的巨噬细胞选择性和特异性表达Cre的小鼠系，并使这种小鼠广泛应用于研究界，将显著影响我们目前研究巨噬细胞的能力，并将对研究巨噬细胞在稳态、免疫和癌症中的研究产生重大影响，从而使该领域向前发展。

DESCRIPTION (provided by applicant): Macrophages are crucial for tissue homeostasis and play essential roles in inflammation, immunity and cancer, and therefore understanding macrophage biology is fundamental for understanding homeostasis and disease. Currently available mice that target Cre expression for the deletion of floxed genes to macrophages have significant and severe restrictions, primarily based on the promoter driving Cre expression. 1) all of the available promoters currently driving Cre in macrophages are active in the entire myeloid lineage and even other cell types, which is frequently stronger than the one in macrophages, and therefore lack macrophage specificity; 2) currently available promoters only target a subset of macrophages; and 3) the two most commonly used Cre lines driven by F4/80 and LysM, are also knocked in one endogenous allele resulting in weak expression and prevent breeding of homozygous mice. The goal of this study is to develop and characterize a novel and improved transgenic mouse line to specifically target expression of Cre to macrophages. We propose to use the human CD68 promoter in combination with a macrophage-specific enhancer to drive expression of an activity-improved Cre and the dtTomato fluorescent protein from a bicistronic transcript for in vivo tracking and sorting. Transgenic mice will be extensively characterized with reporter genes and endogenous floxed genes and compared to the currently available Cre-expressing mice. Therefore our proposal to develop a novel true macrophage selective and specific Cre expressing mouse line in the C57BL/6 strain, and to make this mouse widely available to the research community, will significantly impact our current ability to study macrophages and will have significant ramifications for researchers studying macrophages in homeostasis, immunity and cancer, and will therefore enable the field to progress forward. PUBLIC HEALTH RELEVANCE: Macrophages are essential for tissue homeostasis and contribute to disease. Therefore understanding macrophage biology is fundamental to advance current knowledge of these processes, which requires macrophage specific deletion of genes using the Cre recombinase. Because currently available mice expressing Cre in macrophages have severe disadvantages, our study will develop and characterize a new and substantially improved transgenic mouse with macrophage specific expression of an improved version of Cre, which will be shared with the scientific community.