Figure 4. Schematic describing the generation of Dusp5 ZFN KO rats in the FHH.1^BN genetic background.

Panel A presents the sequence of the Dusp5 ZFN. The Dusp5 specific ZFN introduced a 14 bp deletion and a 3 bp insertion resulting in a net 11 bp deletion between nucleotides 449–464 in the Dusp5 sequence in the KO animals that introduced a frame shift mutation. Panel B presents the strategy for the generation of Dusp5 ZFN KO rats in the FHH.1^BN genetic background. Fertilized donor embryos from female FHH.1^BN rats were collected and the Dusp5 ZFN mRNA was microinjected into pronuclei of the fertilized one-cell embryos. These embryos were transferred back to a foster mother. The heterozygous founders were brother-sister mated to generate the homozygous ZFN KO founders and the FHH.1^BN wild type control rats. Panel C presents an example of PCR genotyping of the region of interest in FHH.1^BN and Dusp5 KO rats. The rats were genotyped using the following primers: Dusp5-F: 5′-GCT GCA GGA GGG CGG CGG CG -3′, Dusp5 R: 5′-CTT TAA GGA AGT AGA CCC G-3′. These primers amplify a 155 bp band for the wild type allele and a 144 bp band for the knockout allele. Both bands are observed in heterozygous rats.