Figure 3
Figure 3. Transgenic analysis of Lmo2 candidate regulatory elements at midgestation. (A) Transgenic mouse embryos at E12.5 showing X-Gal reporter expression in a Lmo2 LacZ knock-in (Lmo2 LacZ KI) and X-Gal reporter expression driven by 3 different LMO2 promoter constructs (pPLacZ, pPexLacZ, and dpLacZ). Whole-mount staining (WM) and representative histologic sections of FL, dorsal aorta (DA), heart (H), yolk sac (YS), and peripheral vessels (V) are depicted. The Lmo2 LacZ KI embryo is macroscopically characterized by staining of vessels, brain, eyes, somites, apical ridges of the limb buds, and tail. Histologically, X-Gal is expressed in peripheral and main vessels, FL, DA, and circulating erythrocytes (see inset at higher magnification of representative areas in FL and H). Compared with the LMO2 349-bp minimal pP (pPLacZ) a 1.3-kb extended version of the pP (pPexLacZ) increases dramatically the expression in endothelial cells. In contrast, the LMO2 dP (dPLacZ) directs expression to the forebrain only (note that the brain section is placed instead of a vessel section). (B) Transgenic mouse embryos at E12.5 showing whole-mount X-Gal staining of the LMO2 minimal pP in collaboration with 1 of 14 putative enhancers. The numbers in the green box of each panel correspond to the distance in kilobases of the putative regulatory elements with respect to the mouse Lmo2 ATG as shown in Figures 1 and 2. Eight enhancer elements −90, −75, −70, −64, −25, −12, +1, and +7 significantly augment the endothelial staining of pPLacZ and/or induced LacZ expression in several additional tissues, such as tail, apical ridges of the limb buds, brain, and potentially FL. In contrast, the elements −58, −47, −43, −40, −35, and −3 show similar or less LacZ expression compared with pP. (C) Comparison of cleared, X-Gal-stained transgenic mouse embryos at E12.5 bearing the Lmo2 LacZ KI (i) and pP combined with the 2 strongest enhancers +1 (ii) and +7 (iii). Besides the strong endothelial enhancement the pP +1 construct shows strong expression in the apical ridges of the limb buds and the tail (ii), whereas pP +7 confers additional brain staining (iii).

Transgenic analysis of Lmo2 candidate regulatory elements at midgestation. (A) Transgenic mouse embryos at E12.5 showing X-Gal reporter expression in a Lmo2 LacZ knock-in (Lmo2 LacZ KI) and X-Gal reporter expression driven by 3 different LMO2 promoter constructs (pPLacZ, pPexLacZ, and dpLacZ). Whole-mount staining (WM) and representative histologic sections of FL, dorsal aorta (DA), heart (H), yolk sac (YS), and peripheral vessels (V) are depicted. The Lmo2 LacZ KI embryo is macroscopically characterized by staining of vessels, brain, eyes, somites, apical ridges of the limb buds, and tail. Histologically, X-Gal is expressed in peripheral and main vessels, FL, DA, and circulating erythrocytes (see inset at higher magnification of representative areas in FL and H). Compared with the LMO2 349-bp minimal pP (pPLacZ) a 1.3-kb extended version of the pP (pPexLacZ) increases dramatically the expression in endothelial cells. In contrast, the LMO2 dP (dPLacZ) directs expression to the forebrain only (note that the brain section is placed instead of a vessel section). (B) Transgenic mouse embryos at E12.5 showing whole-mount X-Gal staining of the LMO2 minimal pP in collaboration with 1 of 14 putative enhancers. The numbers in the green box of each panel correspond to the distance in kilobases of the putative regulatory elements with respect to the mouse Lmo2 ATG as shown in Figures 1 and 2. Eight enhancer elements −90, −75, −70, −64, −25, −12, +1, and +7 significantly augment the endothelial staining of pPLacZ and/or induced LacZ expression in several additional tissues, such as tail, apical ridges of the limb buds, brain, and potentially FL. In contrast, the elements −58, −47, −43, −40, −35, and −3 show similar or less LacZ expression compared with pP. (C) Comparison of cleared, X-Gal-stained transgenic mouse embryos at E12.5 bearing the Lmo2 LacZ KI (i) and pP combined with the 2 strongest enhancers +1 (ii) and +7 (iii). Besides the strong endothelial enhancement the pP +1 construct shows strong expression in the apical ridges of the limb buds and the tail (ii), whereas pP +7 confers additional brain staining (iii).

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