The blood vessel-specific fluorescent transgenic mouse using the Tie2 promoter (Tie2-GFP) has enabled non-invasive real-time in vivo studies of the vascular system. In comparison, a lymphatic-specific fluorescent transgenic mouse model has not yet been established, despite its potential value. Here, we propose the generation of a transgenic mouse line that expresses the red fluorescent protein (RFP) specifically in the lymphatic system. We plan to achieve this goal by undertaking 2 separate approaches. For the first approach, we will utilize binding sites of the lymphatic specific transcription factor Prox1. In the proposal, we present our recent data of characterizing the Proxl-binding sites in the promoter of mouse fibroblast growth factor receptor (FGFR)-3, which we have identified as a Proxl target gene. We demonstrated that the Prox1-response element of 9 nucleotides (CACGCCTCT) is necessary and sufficient for the Prox1-mediated transcriptional activation of a reporter gene. Based on this finding, we will introduce a single or multiple copies of this Prox1-response element into an enhancer/promoter-less RFP reporter vector and microinject this transgene into fertilized mouse oocytes to generate a transgenic animal. In the second approach, we will take an advantage of a recently developed homologous recombination technology to knock-in the RFP gene into the Prox1 loci of 200-bk-long bacterial artificial chromosomes (BACs) that our analysis suggests contain all the regulatory elements needed for the lymphatic-specific expression of Prox1. The resulting Prox1-BAC transgenic reporter constructs will be used to generate the lymphatic-specific RFP mouse line. Furthermore, the lymphatic-specific RFP mouse line will be genetically crossed with the Tie2-GFP mouse line to establish a double transgenic mouse model whose blood and lymphatic vessels are labeled in green and red, respectively. These mouse models will be valuable tools in isolating mouse blood and lymphatic endothelial cells, to study in vivo physiological and pathological angiogenesis and lymphangiogenesis, and to readily visualize the molecular interactions of lymphatic endothelial cells with various immune cells as well as metastatic tumor cells. ? ? ?
