Fig 1.
Generation of Myh6MerCreMer/+ knock-in mouse.
(A) Schematic diagram of gene targeting. The MerCreMer-FRT-Neo-FRT cassette was inserted into the Myh6 locus (6 bp upstream ATG). The Neo cassette was removed by crossing Myh6MerCreMer-Neo/+ mice with the Flippase deleter (FLPe) mice. (B) Long-range PCR of genomic DNA from the positive ES cells generated a 5.9-kb recombinant fragment at 5' end and a 4.3-kb recombinant fragment at 3' end with primers P1/P2 and P3/P4, respectively.
Fig 2.
Myh6-driven Cre recombination is efficiently activated in heart after tamoxifen induction.
X-gal staining revealed robust β-galactosidase expression in the embryonic and postnatal hearts (R26RlacZ/+;Myh6MerCreMer/+) after tamoxifen treatment. No X-gal positive cells were found in other regions on E13.5 embryos (A1). Notched arrows in A2/B1/C1/D1/E1 indicate positive X-gal staining in hearts. Arrowheads in A2/B1/C1/D1/E1 indicate pulmonary artery and aorta. Unnotched arrows in C4/D4/E4 indicate valves. C3/D3/E3 are high magnification images for C2/D2/E2 in the areas labeled by red asterisks. RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; VS, ventricular septum.
Fig 3.
Recombination on Myh6MerCreMer/+ hearts without tamoxifen.
Very few X-gal positive cells were detected on Myh6MerCreMer/+;R26RlacZ/+ hearts without tamoxifen treatment. A is anterior view and B is posterior view of a 4-month-old heart (P120). C and D are high magnification of A and B in the square area, respectively. Arrowheads indicate X-gal positive cell.
Fig 4.
Myh6MerCreMer/+ introduces specific recombination in cardiomyocytes.
R26RtdTomato/+ reporter line was used to trace Myh6 lineage after tamoxifen induction. (A) tdTomato expression on the whole-mount hearts at E13.5-P60. (B-I) Immunostaining on transverse sections of hearts revealed tdTomato expression was co-localized with Tnnt2 (B-E), but was not co-localized with αSMA (F,G) or PECAM (H,I). Notched arrows in B2/C2/D2/E2 indicate tdTomato expression. Arrowheads in B2/C2 indicate lower level expression of tdTomato in VS and ventricular wall. Unnotched arrows in F,G indicate αSMA staining, and in H,I indicate PECAM staining. Arrowheads in C4 indicate valves. B4-G4 are merged images for B1/2/3-G1/2/3, respectively. H2 and I2 are merged images of H1 and I1 with corresponding tdTomato/DAPI images.
Fig 5.
Normal cardiac function and dimensions after tamoxifen induction in Myh6MerCreMer/+ mice.
(A) M-mode echocardiography of the same Myh6MerCreMer/+ and wild type littermate mouse at baseline and five weeks after tamoxifen administration. (B) Quantified indices of left ventricular function and structure before and after Cre induction. Tamoxifen was administered at 0.1 mg/g body weight/day. Ejection fraction, EF; left ventricular internal diameter, LVID; left ventricular anterior wall, LVAW; left ventricular posterior wall, LVPW. All values were plotted as mean ± STDEV.
Fig 6.
Tamoxifen has little effect on Myh6MerCreMer/+ hearts.
Apoptosis and fibrosis assays on Myh6MerCreMer/+ mouse hearts in one week (A-F) and five weeks (G-L) after tamoxifen injection (0.01 mg/g body weight for 3 days). A few apoptotic cells were observed on both R26RtdTomato/+ and Myh6MerCreMer/+;R26RtdTomato/+ hearts after one week (arrows in A,B, P60→P67), and very few detected after five weeks (G,H, P60→P95). Tamoxifen mediates robust recombination on Myh6MerCreMer/+;R26RtdTomato/+ hearts (D,J), but not on R26RtdTomato/+ control hearts (C,I). Myocardial fibrosis was not detected on R26RtdTomato/+ or Myh6MerCreMer/+;R26RtdTomato/+ hearts by trichrome staining after one (E,F) and five weeks (K,L). Image in the upright corner of E is from an unrelated study, showing positive trichrome staining and cardiac fibrosis (arrows). This staining was performed in parallel with sample in E.
Fig 7.
Lower dosage of tamoxifen mediates sufficient recombination on adult Myh6MerCreMer/+ hearts.
Administration of tamoxifen at 0.05 mg/g body weight for 3 days also introduces robust tdTomato expression in Myh6MerCreMer/+;R26RtdTomato/+ mouse hearts (A,B). No recombination occurs in the control hearts (R26RtdTomato/+, C,D). Arrows in A,B indicate coronary vessels.
Table 1.
Transthoracic echocardiography of Myh6MerCreMer/+ mice.