Figure 1. Generation of Alb-hβigh3 transgene

A: The diagram of Alb-hβigh3 transgene shows the albumin enhancer/promoter (Alb E/P), the human βigh3 (hβigh3), and IRES-LacZ-mp1 intron/polyA (IRES-LacZ-mp1/pA). NotI and NruI indicate enzyme sites in the transgene for pronuclear injection. B: PCR genotyping for hβigh3 and LacZ gene in wild-type (wt) and transgenic (tg) mice. The 202-bp hβigh3 and 500 bp LacZ amplification products were produced by PCR in Alb-hβigh3 transgenic mice. PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed as a control. M and F indicate a DNA size marker and a founder mouse as a positive control for PCR, respectively. C: Western blot analysis of hβigh3 expression in liver and eye extracts from wild-type (wt) and transgenic (tg) mice. The hβigh3 protein was highly expressed in the transgenic mouse compared to the wild-type mouse. β-Actin was used as an internal protein control. D: Immunohistochemical study of wild-type (wt) and transgenic (tg) liver and cornea at two months of age using anti-human βigh3 antibody. Expression of hβigh3 transgene was strongly detected in liver hepatocytes and corneal epithelium of Alb-hβigh3 transgenic mouse. Abbreviations: H, hepatocyte; V, terminal hepatic venule; Ep, corneal epithelium; S, corneal stroma; En, corneal endothelium. E: Human βigh3 levels in plasma of wild-type (wt) and transgenic (tg) mice. ELISA showed that hβigh3 levels in plasma were elevated two-fold in transgenic mice. The plasma levels show the average for 19 wild-type mice and 28 transgenic mice. High expression of hβigh3 protein was detected in plasma of transgenic mice by western blotting. The asterisk indicates a statistically significant difference (p<0.001) calculated by paired t-test. The western blotting and ELISA data are representative of three independent experiments.