Generation and characterization of a conditional eNOS knock out mouse model for cell-specific reactivation of eNOS in gain-of-function studies

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) in the vessel wall regulates blood pressure and cardiovascular hemodynamics. In this study, we generated conditional eNOS knock out (KO) mice characterized by a duplicated/inverted exon 2 flanked with two pairs of loxP regions (eNOS^inv/inv); a Cre-recombinase activity induces cell-specific reactivation of eNOS, as a result of a flipping of the inverted exon 2 (eNOS^fl). This work aimed to test the efficiency of the Cre-mediated cell-specific recombination and the resulting eNOS expression/function. As proof of concept, we crossed eNOS^inv/inv mice with DeleterCre^pos (DelCre^pos) mice, expressing Cre recombinase in all cells. We generated heterozygous eNOS^fl/inv or homozygous eNOS^fl/fl mice, and eNOS^inv/inv littermate mice. We found that both eNOS^fl/fl and eNOS^fl/inv mice express eNOS and the overall expression level depends on the number of mutated alleles, while eNOS^inv/inv mice did not show any eNOS expression. Vascular endothelial function was restored in eNOS^fl/fl and eNOS^fl/inv mice, as determined by ACh-dependent vasodilation of aortic rings. Cre-dependent reactivation of eNOS in eNOS^fl/fl and eNOS^fl/inv mice rescued eNOS^inv/inv (phenotypically global eNOS KO) mice from hypertension. These findings demonstrate that eNOS expression is restored in eNOS^fl/fl mice at comparable physiological levels of WT mice, and its functional activity is independent on the number of the reactivated alleles. Therefore, eNOS^inv/inv mice are a useful model for studying the effects of conditional reactivation of eNOS and gene dosage effects in specific cells for gain-of-function studies.