Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice - PubMed (original) (raw)

Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice

T M Schlaeger et al. Proc Natl Acad Sci U S A. 1997.

Abstract

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 beta-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.

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Figures

Figure 3

Figure 3

(A) Structure of the TIE2 locus, including the first exon (shaded box), flanked by upstream and intronic sequences [open box, _Xho_I–_Kpn_I fragment (XK); solid box, _Nco_I–_Xba_I fragment (NcXb)]. (B) Structure of LacZ reporter gene constructs and their respective relative activities (HH− = 100%) in transient transfections of BAECs. Values significantly (P < 0.05; Student’s t test) above 100% have been marked with an asterisk. (C) Activity (in percent of tk core promoter activity) of HH− and HHXK in BAECs and HEK293 cells. (D) Activity of tkXK (tk promoter and 1.7-kb enhancer) and tkNcXb (tk promoter and 303-bp enhancer fragment) in BAECs.

Figure 1

Figure 1

Whole-mount 5-bromo-4-chloro-3-indolyl β-

d

-galactopyranoside-stained embryos at E11.5. (a) An embryo transgenic for the construct HH− shows the reduced staining, as described (4). (b) An embryo transgenic for the construct HHNS shows specific staining in virtually all vessels. TG, number of transgenic embryos analyzed; ES, number showing the endothelial-specific staining shown in the respective picture; ET, number showing ectopic staining; NO, number showing no staining at all.

Figure 2

Figure 2

LacZ staining of an embryo at later embryonic stage (A) and in adult tissues (B). (Aa) In situ hybridization of embryos at E14.5 with LacZ probe. (b) Adjacent section of a, immunostained with anti-PECAM antibody. Identical staining pattern of a and b confirms specific and uniform endothelial expression of LacZ. ∗, Heart; arrow, dorsal aorta. (B) LacZ staining of adult brain (a, b), eye (c), heart (d), kidney medulla (e), kidney cortex (f), intestine (g), and spleen (h, i). In brain (a), virtually all vessels are stained. In higher magnification of the thinner sections of the brain (b), clearly demonstrates the EC staining (arrowheads). In eye (c), uniform and strong LacZ staining of the capillary plexus is indicated by arrowheads. In heart (d), uniform LacZ staining of the myocardial vasculature is evident. LacZ staining was also observed in endocardium (data not shown). In kidney cortex (f), all of the glomerular microvasculatures (arrows) are strongly LacZ-positive. Other microvasculatures are difficult to see with this low magnification unless they are clustered (arrowheads), but higher magnification confirmed the complete staining of all microvasculatures. In intestine (g), LacZ-positive mesenteric vasculature is indicated (arrow). In spleen, low magnification (h) and higher magnification (i) clearly demonstrates the uniform and EC-specific LacZ staining. (Bar = 100 μm.)

Figure 4

Figure 4

Base sequence of the core enhancer. Sequences matching know transcription factor binding site consensus sequences are underlined: CF1 (consensus, ANATGG), αINF2 (AARKGA), GATA (WGATAR), Sp1 (KRGGCKRRK), NF-S (YGTCAGC), Ets1 (SMGGAWGY), CP2-γ (AGCCACT), and PEA3 (AGGAAR); the CACA microsatellite and a putative bZIP transcription factor binding site (see text) are also underlined. Furthermore, the octameric palindrome (see text) is marked by three asterisks.

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