Fig. 3. Generation of Sca-1 KD mice. (A) Construction of the pDECAP-Sca-1 vector. (B,C) Decrease in exogenous Sca-1 expression induced by pDECAP-Sca-1 as shown by RT-PCR (B) and FACS (C) in HEK 293 cells. The total amount (8 µg) of plasmids co-transfected was the same in each experiment (n=3). (D) Decrease in Sca-1 protein levels in the hearts from two independent Sca-1 KD lines. (E) The frequency of CSC colonies from NTG- and Sca-1 KD hearts is shown. Data are expressed as the mean number of colonies formed per 10⁵ single cells deposited ± s.e. (n=4). ^*P<0.01 vs NTG. (F) RT-PCR for embryonic and mesodermal precursor markers. Cardiac fibroblasts were used as negative control. (G) Decrease in Sca-1 expression from two independent Sca-1 KD CSCs clones. Black line, control IgG; red line, Sca-1. (H) Phase-contrast images of respective CSC clones at 14 days of culture in serum-free medium. Bars, 500 µm. (I) Growth kinetics of two independent clonal CSCs isolated from NTG (black lines, C4 and C6) and Sca-1 KD (red lines, C1 and C2) mice. (J) BrdU incorporation, phosphorylated histone-H3 (p-H3) and p53 expression from five independent experiments are shown. ^*P<0.01 vs NTG. (K) Loss of telomerase activity in the clonal CSCs (C1 and C2) isolated from two independent lines of Sca-1 KD mice.