Plasmid construct used to generate R49C
neo gene knock-in mice. The 5' and 3' arms of the αA-crystallin gene (Cryaa) were cloned into a vector containing the floxed neomycin (neo) cDNA. Mutagenesis was performed to mutate amino acid arginine 49 of αA-crystallin to cysteine (R49C). The asterisk above exon 1 indicates the mutation. The numbered blue rectangles indicate exons. The filled triangles are loxP sites and X denotes the XhoI site. Mouse embryonic stem (ES) cells SCC-10 were electroporated with the mutant plasmid, and clones testing positive for neo were identified and used to generate R49Cneo αA-crystallin knock-in mice (WT/R49Cneo). One clone containing wild type (WT) αA-crystallin cDNA and neo was also analyzed and used to generate mice with the neo allele but no mutation (WT/WTneo).