Figure 1. Recombineering strategy used to produce 129S-Cdh23^c.753A and B6-Cdh23^c.753G SNV mice.

To produce B6N strain mice with the Cdh23^c.753G SNV, a targeting vector with DNA from a 129S1/Sv BAC clone was constructed for recombination with C57BL/6 N ES cells (JM8.A3). To produce 129S strain mice with the Cdh23^c.753A SNV, a targeting vector with DNA from a C57BL/6 J BAC clone was constructed for recombination with 129S1/SvImJ ES cells. (A) Targeting vector: The Cdh23 targeted region (9,729 bp) was cloned into a linearized pBlight plasmid (4695 bp) by retrieval from a BAC clone with AB (300 bp) and YZ (320 bp) homology arms. PGK-Neo and LoxP sites then were inserted into the plasmid by retrieval from a synthesized DNA cassette (2320 bp) with CD (200 bp) and EF (203 bp) homology arms. The targeted Cdh23^c.753 SNV (marked by asterisk) is separated from the cassette insertion site by 142 bp and from PGK-Neo by 178 bp. In this figure, the targeted Cdh23^c.753 SNV is shown as the last nucleotide of exon 7 according to Genbank transcript AF308939, which is equivalent to exon 9 in other transcripts such as NM_023370. (B) Wildtype allele: The targeted Cdh23 region of wildtype mouse genomic DNA showing positions of AB and YZ homology arms relative to exons 6 and 7. (C) Knock-in allele: After homologous recombination between the targeting vector and the wildtype Cdh23 allele, ES cells were selected for the presence of the PGK-Neo cassette and the closely linked targeted SNV (asterisk). Restriction enzyme cleavage sites and genomic probes (5′ and 3′) were used to distinguish wildtype and knock-in alleles. (D) The PGK-Neo cassette was subsequently removed by Cre-Lox recombination to produce the final SNV allele.