Fig. 2: Characterization of Kcnq2 cKO and Kcnq2 GOF mouse models.
A Schematic of Kcnq2 wild-type locus and targeting construct containing the inverted mutated exon 4 (mE4). When Cre recombinase is expressed, wild type exon 4 is removed and mE4 exon is inverted to effectively replace wild-type exon 4 with one expressing R201C. We crossed the Kcnq2 floxed line with mice that express Cre under control of the Phox2b promotor to express Kcnq2R201C conditionally (B6(Cg)‐Tg(Phox2b‐cre)3Jke/J::Kcnq2R201C/+; Kcnq2 GOF). B Genotyping PCR analysis for Kcnq2 GOF and Kcnq2 cKO mice. The PCR products run to the expected sizes for each genotype and primer set (Kcnq2 GOF primers span exon 4, including residue 201 of exon 4; Kcnq2 cKO primers include a loxP site). Water was used as a no-template negative control. C, Immunohistochemistry was performed to characterize expression of Kcnq2 protein in Phox2b-expressing ventral parafacial neurons in brainstem sections from Kcnq2+/+, Kcnq2 GOF, and Kcnq2 cKO mice. Images of coronal medullary sections (~6.24 mm behind bregma) from Kcnq2+/+ and Kcnq2 GOF mice show robust Kcnq2 signal (green) co-localized with Phox2b labeling (red); and confirm reduction of Kcnq2 protein in Phox2b-expressing ventral parafacial neurons in sections from Kcnq2 cKO mice. Right, summary of immunohistochemistry results shows the relative proportions of Phox2b-expressing neurons in the ventral parafacial region is comparable between Kcnq2+/+ (N = 3 animals; n = 1240 cells), Kcnq2 GOF (N = 3 animals, n = 956 cells), and Kcnq2 cKO (N = 2 animals, n = 486 cells) mice. We also found similar proportions of Phox2b-expressing cells in sections from Kcnq2+/+ (59%) and Kcnq2 GOF (57%) mice are Kcnq2-immunoreactive (Kcnq2-IR), whereas approximately a quarter of Phox2b-expressing cells did not show detectable levels of Kcnq2, and a relatively modest level of Kcnq2-IR was also observed in Phox2b-negative cells. As expected, virtually all (96%) Phox2b-expressing neurons in the ventral parafacial region in sections from Kcnq2 cKO mice lack expression of Kcnq2. Scale bar 50 µm. D survival curves for each experimental group as well as floxed-only control mice (Phox2b+/+::Kcnq2R201C/+) show that each genotype exhibits normal survival. These results were compared using Kaplan-Meier survival curve comparison.
