Control lentiviral plko 1 vectors

Control lentiviral pLKO.1 vectors and pLKO.1 vectors containing shRNAs for mouse c-Abl were obtained from Thermo Fisher Scientific^{44 (link),45 (link),48 (link)}. The shRNA sequences of mouse c-Abl included clone numbers 54 (5′-AATACTCCAAA TGCCCACACG-3′), 55 (5′-AAGGTGGAT GAGTCAAAC TGC-3′), 56 (5′-TAGCTCACA CAGAAAGTG TAC-3′), 57 (5′-AACAGGTTG GTCTTTCT GTCT-3′), and 58 (5′-TACTTC TTCCAAACGCCC TCG-3′). Viral particles were produced as described above. To establish hematopoietic cells with stable c-Abl knockdown, Lin⁻ cells from E14.5 Hem-1^+/+ embryos were maintained in Stemspan medium with TPO (10 ng/ml) and SCF (10 ng/ml) and infected twice with viral particles containing pLKO.1-c-Abl shRNA or pLKO.1-CTL shRNA under centrifugation (900 × g) at 32 °C for 30 min. Stably transduced cells were selected with puromycin (2 mg/ml) for 2 days. The infected cells were transplanted into 9.5 Gy lethally irradiated CD45.1 recipient mice along with 5 × 10⁵ BMCs from the recipient mice. The analysis of engraftment in the peripheral blood was performed monthly after transplantation. At 4 months post transplantation, the BMCs were used to analyze the engraftment ability. c-Abl knockdown using different shRNA clones in Lin⁻ cells was determined by western blotting as shown in Supplementary Fig. 9e.