Generation of a Cre-LoxP mouse line expressing hCXCR1

描述(申请人提供)：这个项目的总体目标是建立一个以Cre重组酶驱动的组织特异性方式表达人CXCR1基因的转基因小鼠系，作为研究CXCR1依赖的趋化因子功能的工具。趋化因子是在生理和病理条件下发挥多种作用的重要分子，IL-8是目前研究最多的趋化因子之一，因其在炎症、伤口愈合、血管生成和肿瘤发生等方面发挥重要作用。为了阐明这种趋化因子的功能，已经做了很多工作，但由于缺乏可以通过基因操纵的动物模型系统，进展受到了阻碍。在人类中，IL-8与两个受体CXCR1和CXCR2相互作用，因此，为了确定IL-8如何发挥作用，有必要研究这两个受体在HLL-8激活后所起的作用。这些受体和r|L-8与人类蛋白高度同源，兔表达这两种受体。然而，在兔子身上进行基因操作是非常困难的(如果可能的话)和昂贵的。另一方面，小鼠没有CXCR1基因或IL-8，因此不能用来全面研究IL-8在人类中的功能。我们在这里提出了一项计划，建立一个人CXCR1转基因小鼠系，作为体内实验系统，以阐明IL-8的功能。这项工作的具体目标是：1)构建和测试转基因动物的DMA构建。2)获得方正转基因小鼠。3)检测转基因小鼠的组织特异性Cre重组酶活性。我们将首先建立一个基于loxP的条件转基因方块系，其中hCXCRI在小鼠所有组织中的表达都被抑制。为此，将通过分子克隆的方法制备DNA转基因构建物，并在细胞培养系统中进行测试，以确认其功能，包括转基因hCXCRI的表达和Cre-重组酶介导的重组。然后，我们将向异源生物提供DNA转基因构建物，以创造出方正转基因小鼠。最后，我们将获得特异性表达Cre重组酶的小鼠，并将它们与我们的创始小鼠杂交，实现hCXCRI转基因在皮肤中的激活，以测试小鼠转基因品系的体内功能。在特定组织中表达hCXCRI的小鼠将成为进行与人白细胞介素8相关研究的有力工具，因为交付的hIL-8将能够与相关组织中的hCXCRI和mCXCR2(已被证明发生)相互作用，从而深入了解IL-8在人类中的作用的分子机制。总的来说，我们提议建立的转基因动物将是一个有用的工具，它将为我们的研究和其他人的研究带来多功能性，以了解HLL-8的功能。

DESCRIPTION (provided by applicant): The overall objective of this project is to generate a transgenic mouse line that expresses the human CXCR1 gene in a Cre-recombinase-driven tissue-specific manner as a tool to study CXCR1-dependent chemokine functions. Chemokines are important molecules playing many roles in physiological and pathological conditions, lnterteukine-8 is one of the chemokines that has been intensively studied because it functions in inflammation, wound healing, angiogenesis and tumorigenesis. Much work has been performed to elucidate the functions of this chemokine but progress has been impaired by the lack of an animal model system that can be genetically manipulated. In humans, IL-8 interacts with two receptors, CXCR1 and CXCR2, hence in order to determine how IL-8 functions it is necessary to study the role of each of these receptors in response to activation by hlL-8. These receptors and r|L-8 are highly homologous to the human proteins and rabbits express both receptors. However, it is very difficult (if at all possible) and expensive to perform gene manipulations in rabbits. Mice, on the other hand, do not have the CXCR1 gene or IL-8, hence cannot be used to fully study the functions of IL-8 in humans. We propose here a plan to create a human CXCR1 transgenic mouse line that can serve as an in vivo experimental system to elucidate the functions of IL-8. The specific aims of the work are: 1) To build and test the DMA construct for the transgenic animal. 2) To produce the founder transgenic mice. 3) To test the transgenic mouse line for tissue-specific Cre- recombinase activation. We will first generate a LoxP-based conditional transgenic founder line in which hCXCRI expression is suppressed in all tissues of the mouse. To do so, a DNA transgenic construct will be prepared by molecular cloning methods, tested in a cell culture system to confirm its functionality, including the transgene hCXCRI expression, and Cre-recombinase mediated recombination. We will then supply the DNA transgenic construct to Xenogen Biological that will create the founder transgenic mice. Finally, we will acquire specific Cre-recombinase expressing mice and cross them with our founder mice to achieve hCXCRI transgene activation in the skin to test the functionality of the mouse transgenic line in vivo. The mice expressing hCXCRI in specific tissues will serve as powerful tools to perform research related to human interieukin-8 because delivered hlL-8 will be able to interact with hCXCRI and mCXCR2 (already shown to occur) in the pertinent tissues, providing insight into the molecular mechanisms of the functions of IL-8 in humans. In general, the transgenic animal we are proposing to build will be a useful tool that brings versatility to our research and those of others for understanding the functions of hlL-8.