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

描述（由申请人提供）：巨噬细胞对于组织稳态至关重要，并在炎症、免疫和癌症中发挥重要作用，因此理解巨噬细胞生物学对于理解稳态和疾病至关重要。目前可获得的靶向Cre表达以使floxed基因缺失至巨噬细胞的小鼠具有显著且严重的限制，主要基于驱动Cre表达的启动子。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它细胞类型中都有活性，其通常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靶向巨噬细胞的亚群;和3）由F4/80和LysM驱动的两个最常用的Cre系也被敲入一个内源等位基因，导致弱表达并阻止纯合小鼠的繁殖。本研究的目的是开发和表征一种新的和改进的转基因小鼠系，以特异性靶向表达Cre的巨噬细胞。我们建议使用人CD 68启动子与巨噬细胞特异性增强子组合，以驱动活性改善的Cre和dtTomato荧光蛋白从双顺反子转录物的表达，用于体内跟踪和分选。转基因小鼠将用报告基因和内源性floxed基因进行广泛表征，并与目前可用的Cre表达小鼠进行比较。因此，我们提出在C57 BL/6品系中开发一种新的真正的巨噬细胞选择性和特异性Cre表达小鼠系，并使这种小鼠广泛用于研究界，这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稳态，免疫和癌症的研究人员产生重大影响，因此将使该领域向前发展。 公共卫生相关性：巨噬细胞对组织稳态至关重要，并有助于疾病。因此，理解巨噬细胞生物学是推进这些过程的现有知识的基础，这需要使用Cre重组酶的巨噬细胞特异性基因缺失。由于目前可用的小鼠表达Cre在巨噬细胞有严重的缺点，我们的研究将开发和表征一种新的和大幅改善的转基因小鼠与巨噬细胞特异性表达的Cre的改进版本，这将与科学界共享。

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.