mice by the hot hydroxide method, and genotyping was performed with the same PCR primers and cycling conditions used for the CEL I nuclease assay. Positive founders were identified by Sanger sequencing of PCR products. The single positive founder was bred to a wild-type C57BL/6J mouse, and the resulting progeny were intercrossed for one to two generations to breed the knock-in allele to homozygosity. Genotyping of progeny was performed in the same manner. Wild-type and homozygous knock-in littermates of both sexes from several litters, ~12 weeks of age, were used for gene expression analyses.
