Fig. 1. Generation of RET S697A mutant mice. (A) The S697A mutation introduced in mouse RET exon 11. (B) Sequencing of amplified PCR product containing the S697A mutation site. Template DNA was prepared from clone 106 shown in (D). (C) Schematic representation of the targeting strategy. The S697A mutation was introduced in RET exon 11 and the loxP flanked neo gene in intron 11 in the targeting vector. After homologous recombination in ES cells, RET^S697A-neo mutant mice were generated. The neo gene was removed to generate RET^S697A mutant mice by crossing with ß-actin promoter/Cre transgenic mice. P1 and P2 indicate the forward and reverse primers for genotyping. H, HindIII; B, BamHI. (D) Southern blot analysis for RET^S697A-neo and RET^S697A mutations. Genomic DNA was prepared from control W9.5 ES cells (W9.5), homologous recombinant clone (clone 106), and tails of wild-type (Wt) and heterozygous RET S697A mutant mice (RET^S697A/+). DNA was digested with HindIII, and analyzed with the indicated 5' external probe shown in (C). Wild-type and RET^S697A-neo mutant alleles generate 9.0 kb and 4.8 kb bands, respectively. Loss of 4.8 kb band in DNA from a RET^S697A/+ mutant mouse indicates successful removal of the neo gene. (E) Genotyping of mutant mice. P1 and P2 primers shown in (B) were used for genotyping by PCR. As the S697A mutant allele contains a 70 bp insertion, wild-type and mutant alleles give 302 bp and 372 bp bands, respectively. (F) RET protein expression in mutant mice. Lysates prepared from whole embryos at E11.5, newborn brain stems and stomachs were subjected to western blotting with anti-RET antibody. The 170 kDa and 150 kDa RET proteins are indicated. ß-Actin expression was investigated as a loading control. (G) Phosphorylation of Serine 697 in DRG neurons. Lysates were prepared from cultured DRG neurons isolated from wild-type and mutant mice, and subjected to western blotting with anti-RET and anti-phospho-RET(S697) antibodies (left and middle panels). The membrane was treated with alkaline phosphatase (AP) before incubation with anti-phospho-RET(S697) antibody to confirm that the detected band represents the phosphorylated protein (right panel).