Fig. 1.

Screening animals for zinc finger nuclease (ZFN)-targeted mutation at the regulated endocrine-specific protein 18 (Resp18) locus. A: tail DNA samples from pups born postmicroinjection of custom ZFNs targeting exon 3 of the Resp18 locus were screened by PCR amplification with primers designed to amplify rat genomic fragments encompassing the ZFN-targeted site. Representative sequencing results from the PCR products shown with a 7 bp deletion in the mutant rat’s highlighted in blue box. Also represented are the corresponding translated peptide sequences, which were altered as a result of the 7 bp nucleotide deletion. B: real-time PCR analysis of Resp18 mRNA expression in heart and kidney samples from Resp18^mutant and Dahl salt-sensitive (SS) rats fed on low- and high-salt diet. mRNA obtained from heart and kidney were converted to cDNA and quantified with gene-specific primers. Gapdh was used as internal control. Values are fold change in SS (n = 6), Resp18^mutant (n = 6); Data are means ± SE. Bars labeled with different letters were significantly different from each other; levels of statistical significance were analyzed by one-way ANOVA *P ≤ 0.05. LS, low salt; HS, high salt. C: Western blot analysis of Resp18 protein expression in SS and Resp18^mutant rat kidney lysates. The anti-resp18 polyclonal antibody (JH1162) was a gift from Prof. Betty Eipper; anti-Gapdh antibody (#3683) was purchased from Cell Signaling.