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. ? ?
|Baquerizo Nole, Katherine L; Yim, Elizabeth; Van Driessche, Freya et al. (2014) Wound research funding from alternative sources of federal funds in 2012. Wound Repair Regen 22:295-300|
|Njauw, Ching-Ni; Yuan, Hongwei; Zheng, Lei et al. (2008) Origin of periendothelial cells in microvessels derived from human microvascular endothelial cells. Int J Biochem Cell Biol 40:710-20|
|Zheng, Lei; Njauw, Ching-Ni; Martins-Green, Manuela (2007) A hCXCR1 transgenic mouse model containing a conditional color-switching system for imaging of hCXCL8/IL-8 functions in vivo. J Leukoc Biol 82:1247-56|