Figure 6
Generation of transgenic mice and expression of the EGFP reporter gene. (A) The quality and yield of the purified transgenes for microinjection were confirmed by CHEF. All signals indicate intact fragments and sufficient amounts for microinjection. The concentration of retrieved transgenes ranged from 0.3 to 3 ng/μl. (B) Schematic diagram of Tg-110 and Tg-250SB. Tg-250SB transgenic mice express the EGFP reporter gene, whereas Tg-110 transgenic mice do not. The number of EGFP-positive independent founders and lines among the total analyzed is shown in parentheses. (C) Whole-mount images of the medial aspect of the OB in transgenic Tg-110 (line #10, left panel) and Tg-250SB mice (line #8, medial panel) and the MOR42-1-iTG [28] gene-targeted mouse (right panel). Tg-110 transgenic mice do not show EGFP fluorescence, whereas Tg-250SB transgenic mice do. EGFP-labeled axons in Tg-250SB converge on the dorsal side of the OB and form a glomerulus. Arrowheads indicate glomeruli. D, dorsal; V, ventral; A, anterior; P, posterior. (D) Southern blot analysis of Tg-250SB line #8 and its transgene-negative littermate (control). The MOR42-3 probe detected the endogenous (2.0 kb, open arrowhead) and the transgenic (1.2 kb, closed arrowhead) MOR42-3 genes, and also detect both endogenous and transgenic MOR42-1 gene (13.6 kb, arrow). (E) Confocal images of Tg-250SB line #8 transgenic and MOR42-1-iTG gene-targeted mice. Mosaic patterns of EGFP-expressing OSNs in the dorsal epithelium (medial view, left panel) and glomeruli formed by axons in the OB (dorsal view, right panel) were observed. (F) Tg-MOR42-3 (left panel) or endogenous-MOR42-1 glomeruli (right panel) were detected by immunostaining coronal cryosections. Red, OMP immunoreactivity. Green, EGFP immunoreactivity. Blue, DAPI nuclear staining, which marks the glomeruli structure. The glomeruli in the Tg-250SB transgenic mouse showed an intermingling of red (EGFP-negative) and yellow (EGFP-positive) axons, whereas the glomeruli in the homozygous MOR42-1-iTG mouse were fully doubly labeled. Scale bars: E, 500 μm; F, 30 μm.