Figure 1. A Gata2 vascular enhancer confers CreERT2 and mCh transgene expression in the embryonic vasculature.
(A) Schematic depicting the CreERT2 (15) or mCh (19) cDNA driven by the HSV TK promoter (tkP) and the 1.2-kbp Gata2 VE (5) in VECreERT2 or VEmCherry expression plasmid, respectively. Each minigene cassette was flanked by tandem repeats of chicken HS4 insulators (ins) (53). Both inserts were excised from the vector and coinjected (1:1) into the pronuclei of mouse oocytes to generate doubly transgenic (TgVE) mice. F2–F5 progeny were used in subsequent analyses (B–E). (B) Cre transgene copy number (normalized to Actin) was determined by qPCR to range between 5 and 47 copies. Cre mRNA level (normalized to endogenous endothelia-restricted Flk1 mRNA) in the heart (black bars) and kidney (white bars) of neonatal TgVE pups (n = 3 to 8) was determined by RT-qPCR. Of the 3 TgVE lines that showed significant endothelial mCh staining (see below), TgVE56 and TgVE62 both robustly expressed Cre mRNA, while Cre transcripts were barely detectable in TgVE73. qPCR primer sequences are listed in Supplemental Table 1. Data represent mean ± SD. (C) mCh epifluorescence in representative E10.5 embryos. Of 7 lines that stably transmitted both transgenes, TgVE62 expressed mCh most robustly in an endothelia-specific manner, while in some lines mCh was weakly expressed (e.g., TgVE60 and TgVE473). (D) Coincident expression (merge) of mCh (TgVE62) and eGFP (generated from Gata2+/gfp; ref. 21) in the major and fine cranial vasculature in an E10.5 TgVE62:Gata2+/gfp compound mutant embryo. mCh expression temporally and spatially parallels that of eGFP (Gata2) in the vasculature. The asterisk indicates a GFP-exclusive area of Gata2 expression in the ventral midbrain. (E) Coincidence (merge) of mCh (TgVE62) and eGFP (from TgTie2.gfp) epifluorescence in the major and intersomitic vasculature of an E10.5 TgVE62:TgTie2.gfp embryo, underscoring the vascular endothelial tissue specificity of the TgVE transgene.