(A) Resultant murine Gne (Uea1) genomic locus, exons 11 and 12, after homologous recombination with the sequence-verified targeting vector. The M712T mutation was created in exon 12, and a neo cassette (under the PGK promoter) flanked by flippase recombinase target (FRT) sites was inserted. LoxP sites were inserted before exon 12 and after the PGK-neo gene. (B) Genotyping of mutant mice. A PCR-amplified 387-bp fragment of genomic DNA across the M712T (ATG to ACG) mutation was digested by the NlaIII restriction endonuclease into 265-bp, 89-bp, and 33-bp fragments in a wild-type allele (+) and into 354-bp and 33-bp fragments in a mutant M712T allele (–). c, cut; MW, molecular weight; u, uncut. (C) RT-PCR of kidney and skeletal muscle RNA. RNA was reverse transcribed and PCR-amplified using primers covering exons 11 and 12 (355 bp). Digestion by NlaIII cut the wild-type allele (+) into 225-bp, 89-bp, and 41-bp fragments and the mutant M712T allele (–) into 314-bp and 41-bp fragments. Digestion confirmed the exclusive presence of the mutant M712T allele in Gne^M712T/M712T (–/–) tissues. (D) Numbers of mice at E17–E19 and P21. At P21, genotyping of 76 mice from 13 litters (9 Gne^M712T/+ matings) identified only 1 Gne^M712T/M712T offspring. Subsequent genotyping of 35 E17–E19 embryos from 4 Gne^M712T/+ mice yielded a Mendelian distribution of genotypes. (E) At P2, Gne^M712T/M712T pups were smaller than their heterozygous (Gne^M712T/+) and wild-type (Gne^+/+) littermates and lacked a prominent milkspot.