Figure 1.

Generation of the D477G KI mouse model. A: The D477G targeting strategy is shown as a schematic representation. The targeting construct is designed to span 5.7 kb upstream of the point mutation (LA) and 1.7 kb downstream of the Neo cassette (RA). The Neo cassette was inserted in the intron region between exons 13 and 14. FRT-flanked Neo cassette in the targeted allele is deleted by FRT-FLP recombination, thereby generating a mutant allele containing the point mutation in exon 13 (asterisks). B: A PCR strategy was used to screen for the positive clones. LAN1 and A2 primer pair targeting the RA yielded a 2.18-kb product. WT genomic DNA was used as a negative control, and an individual clone (before reconfirmation) was used as a positive control. C: The PCR-confirmed clones were further validated by Southern blotting. DNA digested with ScaI or MfeI was hybridized with a probe against the Neo cassette. Corresponding signals from ScaI and MfeI were detected at 12.5 and 9 kb, respectively. D: Genotyping PCR using primers F1 and R1 flanking the FRT site after Flp-mediated deletion of the Neo cassette produced 549- and 448-bp products, each representing targeted and WT allele, respectively. Extra 101 bp present in targeted allele comprises 34-bp-long FRT sequence and 67-bp-long remnants from the Neo cassette. E: Sequence confirmation of the point mutation at codon 477. c.1430G>A is confirmed by sequencing the PCR amplicon obtained with a primer pair encompassing the region of the point mutation. +Ve, positive; F, forward; HET, heterozygous; HYB, hybrid; KI, knock in; LA, left homology arm; R, reverse; RA, right homology arm; WT, wild type.