FIGURE 3.

Establishment of Neu5Gc-overexpressing Cmah Tg mice. A, construct for Cre-inducible transgenic Cmah expression. In the presence of Cre recombinase, the floxed EGFP gene is eliminated, and the Cmah gene is expressed under a strong CAG promoter. B, Neu5Gc expression in nestin-Cre-positive Cmah Tg mice (NCmahTg). Neu5Gc% in total sialic acids of brain lysate was determined by acid release, DMB derivatization, and HPLC. Each bar represents result obtained from an independent animal. C, transgene-derived Cmah expression in NCmahTg mice. Cmah expression was examined by Western blotting. D, Neu5Gc incorporation into various proteins. Neu5Gc-containing sialoglycoproteins were detected by Western blotting using chicken anti-Neu5Gc IgY. E, immunohistochemical detection of Neu5Gc in the brain. Frozen brain sections were stained with chicken anti-Neu5Gc IgY and visualized with HRP substrate (red). Nuclei were counterstained with Mayer's hematoxylin (blue). Scale bar = 100 μm. F, birth ratios of transgenic mice. The ratios of wild-type (WT), Cmah transgene only (EGFP(+)), Cre only (Cre(+)), and Cmah transgene-positive Cre-positive NCmahTg (Tg) mice among a total of 741 mice are shown. G, brains from 6-month-old mice after perfusion-fixation. Scale bar = 5 mm. WT, wild-type; Cre, Cre-positive (no Cmah transgene); Tg, NCmahTg. Data are representative of multiple independent experiments that yielded similar results.