Figure 1. Generation and characterization of BAC hOPTN^E50K transgenic mice.

(A) BAC transgenic mice expressing disease-associated mutant E50K human optineurin (BAC hOPTN^E50K) were generated by using genomic sequences coding for the optineurin promoter region and the human OPTN locus. (B) Quantification of human optineurin mRNA in 18-month old BAC hOPTN^E50K (E50K) and wildtype (WT) mice. E50K hOPTN overexpression was observed in line I, but not line II (E50K I, E50K II). Mouse OPTN (mOPTN) transcripts were similar in all three genotypes (n = 3 for each genotypes). Data presented as mean ± S.E.M. (C) Optineurin protein expression in isolated retinas from 18 month old mice were analyzed by immunoblot analysis. Shown are two different animals for each genotype. Using an antibody that recognizes human optineurin but not mouse optineurin, the expression of the BAC transgene was observed in retinas from line I of BAC hOPTN^E50K mice.

(D) In young, 2-3 month old E50K and WT mice (n = 5 per genotype), the IOP was similar between the two gentoypes (p = 0.77) and between the right (OD) and left (OS) eyes within each genotype (p = 0.39 for E50K; p = 0.88 for WT). Similarly, in aged 18-month old E50K (n = 11) and WT (n = 10) mice, the IOP was not different between different genotypes (p = 0.05) or between the right and left eyes within each genotype (p = 0.39 for E50K; p = 0.20 for WT). (E) hOPTN expression pattern is similar to endogenous mouse OPTN. Immunofluorescent labeling in retina cryosections from 18-month old wildtype (WT) or BAC-hOPTN^E50K (E50K) mice. Brn3a was used as a marker for retinal ganglion cells. Using an antibody specific for human optineurin (hOPTN), immunoreactivity was observed only in BAC-hOPTN^E50K but not wildtype retinas (top panels). Mouse OPTN expression was observed with a different antibody that recognizes both human and mouse optineurin (bottom panels). n=3 animals per genotype. Scale bars 50 m.