Figure 7.
Generation of Clrn1N48K knock-in mouse. A, Targeting map for Clrn1N48K knock-in mice. Top, Mouse Clrn1 consists of four exons. Second row, The targeting vector contained 5.7 kb of 5′ and 2.2 kb of 3′ homologous sequence with C-to-G transversion at the codon 48. Third row, As a result of homologous recombination, codon 48 in exon 1 was changed to AAG, and the loxP flanked Neo gene was inserted after exon 1. Bottom row, Neo was removed from targeted locus by cre–loxP recombination. B, DNA sequence analysis confirms mutation at the target nucleotide in exon 1: both wild-type nucleotide C and mutant nucleotide G were observed in a cDNA sample from heterozygous knock-in mice (+/KI). C, The missense mutation introduced a BsaHI restriction site, which was used to distinguish the wild-type (+/+), heterozygous (+/KI), and homozygous (KI/KI) mutants. D, Mouse Clrn1 mRNA is expressed in brain, cochlea, and retina; the upper band (∼850 bp) and the lower band (∼750 bp) seen in the upper half is typical Clrn1 mRNA and alternative splicing expression pattern. Results show that Clrn1 mRNA expression is not affected by the missense mutation. WT, Wild type.