TIE2 is a vascular endothelial-specific receptor tyrosine kinase essential for the regulation of vascular network formation and remodeling. Previously, we have shown that the 1.2-kb 5 flanking region of the TIE2 promoter is capable of directing -galactosidase reporter gene expression specifically into a subset of endothelial cells (ECs) of transgenic mouse embryos. However, transgene activity was restricted to early embryonic stages and not detectable in adult mice. Herein we describe the identification and characterization of an autonomous endothelial-specific enhancer in the first intron of the mouse TIE2 gene. Furthermore, combination of the TIE2 promoter with an intron fragment containing this enhancer allows it to target reporter gene expression specifically and uniformly to virtually all vascular ECs throughout embryogenesis and adulthood. To our knowledge, this is the first time that an in vivo expression system has been assembled by which heterologous genes can be targeted exclusively to the ECs of the entire vasculature. This should be a valuable tool to address the function of genes during physiological and pathological processes of vascular ECs in vivo. Furthermore, we were able to identify a short region critical for enhancer function in vivo that contains putative binding sites for Ets-like transcription factors. This should, therefore, allow us to determine the molecular mechanisms underlying the vascular-EC-specific expression of the TIE2 gene.Establishment of the circulation is critical for the development of all organ systems of the human body, and endothelial cells (ECs), which line the lumina of all blood vessels, play an essential role in the formation and physiological functions of the circulatory system (for reviews, see refs. 1 and 2). In addition, the importance of the vascular system during pathological conditions such as cancer, atherosclerosis, and wound healing has also been well recognized (for review, see ref.3). However, little is known about the molecular mechanisms regulating the diverse functions of ECs in vivo.The recent development of transgenic mouse technology has proven to be a powerful tool for the examination of a number of mammalian developmental processes, and sequences that are able to drive gene expression in ECs of transgenic mice have been described (4-7). However, none of these sequences work uniformly in all ECs of all developmental stages or in the adult animal. Furthermore, some of these expression systems are not strictly EC-specific (5-7). Therefore, we have embarked on the development of an in vivo expression system in which heterologous gene expression can be targeted specifically and uniformly to ECs throughout development and the adult animal.The TIE2 gene, coding for one of the receptors of the newly identified family of regulators of vascular remodeling, called the angiopoietins (8, 9), appeared to be a promising candidate in the search for EC-specific transcriptional regulatory elements. This is because TIE2 expression becomes detectable as soo...
