Figure 1.

Generation of AMELX-S16A knockin mice. A, schematic diagram of WT AMELX locus, targeted and neomycin resistance cassette-deleted alleles. Exon 3 was replaced with the knockin construct containing the S16A point mutation (black bar) with FRT-flanked neomycin selection cassette. FRT sites are represented by gray triangles. Not drawn to scale. B, PCR using F1/R1 primers amplify the 2.60-kb fragment. ES clones 1–4 were identified as positive and selected for expansion. DNA from an individual clone and no DNA were used as positive (+) and negative (−) controls, respectively. Confirmation of the point mutation was performed by PCR using F2/R2 primers. This reaction produces a product 0.99 kb in size. Primer set F3/R3 was used to screen F1 female mice for the deletion of the Neo cassette. The PCR product for the WT is 254 bp and for the Neo-deleted, 289 bp. Heterozygous female mice were set up to mate with WT C57BL/6 males to generate offspring.