Fig. 1. Ret^DN/+ mice fail to thrive. (A) Mutant or wild-type human RET9 cDNA was introduced into exon 1 (black box) of the mouse Ret locus (black triangles, loxP sites). The restriction sites (K, KpnI; N, NcoI) and length of the expected fragments in Southern blots are indicated. (B) Schematic of Ret knocked-in alleles. Open box, RET9 cDNA; black line, SV40 3'UTR; ECD, extracellular domain; TK, tyrosine kinase domain; triangle, loxP sites; L985P and Y1062F, RET mutations in the DN allele. (C) Southern blot analysis confirmed successful generation of Ret mutant mouse lines. (D) Growth curves of Ret^DN/+ and wild-type (wt) littermates at indicated postnatal time points (n=3 for each time point; ^*=P<0.01; error bars, mean±s.d.). (E) Photograph showing the typical size difference between 3-week-old Ret^DN/+ and wild-type littermates. (F) Expression of Ret mutant alleles in mouse brain RNA (P0) by RT-PCR. Total, endogenous mouse and knocked-in human RET; knockin, mice harboring the indicated human RET cDNA (Ret9 or RetDN) in the Ret locus generate a knockin-specific product; knockout, Ret^TGM mice where the ret locus is inactivated with the TGM allele. Note that retention of the neomycin resistance gene (Ret^DNneo/+) results in a marked reduction in expression of the knocked-in RetDN allele. wt, wild type. cDNAs knocked-in in the ret locus: RetDN, mutant human RET9 (L985P, Y1062F); Ret9, human wild-type RET9; RetTGM, tauEGFP-myc; RetDNneo, retained neomycin resistance gene in RetDN allele. `+' denotes one copy of the endogenous mouse allele.