Fig. 1
Generation of rats with Bmpr2 mutation using CRISPR/Cas9. A Wild-type (WT) Bmpr2 gene. Guide RNA (gRNA) was designed to target the sequence indicated by underline next to protospacer adjacent motif (PAM, GGG indicated by dashed underline). (Top right) The electropherograms with single waveforms in WT rat. B A single nucleotide (guanine) insertion at position 43–44 (G in bold and underline) in the exon 1 of BMPR2 coding DNA (designated as “44insG”), causes a frameshift at amino acid 15 (Leu15Cys) and results into Bmpr2 translation termination (TAA indicated by underline) at amino acid 37 (22 amino acid down from the first frameshift). (Bottom right) The electropherograms with double waveforms after the site of mutation in a +/44insG rat. C, D Dysregulated BMPR2 signalling in the lung of the +/44insG rats at 7 weeks of age. Representative Western blots and the quantification of relative expression of BMPR2 protein (C, n = 4 for each group) and its downstream substrate proteins SMAD1/5/9 (n = 3 for each group), phosphorylated SMAD1/5/9 (n = 3 for each group) and the pSMAD/SMAD1/5/9 ratio (D). β-actin was used as the loading control. E–I Schematic illustration of the study protocols. Seven-week-old rats were injected with monocrotaline (MCT, 60 mg/kg) or saline as indicated. E Assessment at 3 weeks after MCT or saline injection in both male and female +/44insG and WT rats (n = 43 for male and n = 23 for females). F Survival study in male rats, 4 weeks following MCT injection in both +/44insG and WT littermates without treatment (n = 32). G Survival with a phosphodiesterase type 5 inhibitor, tadalafil (10 mg/kg) therapy from day 14 to day 28 (n = 18) and from day 14 to day 42 (n = 27) after MCT injection in male +/44insG and WT littermates. H Natural course of male +/44insG and WT littermates at 6 months of age. I Chronic hypoxia model for male +/44insG and WT littermates (n = 11). Evaluation was done after 3 weeks of exposure to hypobaric hypoxia (380 mmHg). Data are presented as means ± SEM; unpaired t-test; *p < 0.05, ***p < 0.001. Numbers 1–13 = Exon numbers; gRNA = guide RNA; PAM = protospacer adjacent motif; A = adenine; G = guanine; C = cytosine; T = thymine; BMPR2 = bone morphogenetic protein receptor type 2; WT = wild-type; +/44insG = Bmpr2 mutant rat; MCT = monocrotaline;
= female rats
= male rats