Lenti zip mir 22

For construction of the recombinant lentiviruses that expresses specific shRNAs against MECOM or PU.1, the targeted sequences (see S2 Table) were synthesized and inserted into the pLentiLox 3.7-RNAi plasmid (Invitrogen) following the manufacturer’s protocols. For construction of the recombinant lentivirus that expresses miR-22, a 300-bp DNA fragment containing the miR-22 precursor was amplified and inserted into pMiRNA1 vector. The miRZip lentivector construct expressing miRZip shRNAs targeting miR-22 (Lenti-ZIP-miR-22) was purchased from SBI (Mountain View, CA, USA). The virus packaging was performed using a packaging kit from SBI (Mountain View) according to the manufacturer’s instructions. The lentivirus particles (Lenti-miR-22, Lenti-Con, Lenti-ZIP-miR-22, Lenti-ZIP-Con, shMECOM, shPU.1, shCon) were harvested and concentrated using PEG-it Virus Precipitation Solution (SBI). The lentiviral particles were added into the THP1 cells or CD34⁺ cells in the presence of Polybrene (5 μg/mL; Sigma, St. Louis, MO, USA). The cells were washed with PBS 24 hours after infection and exposed to lineage-specific differentiation cultures or plated for colony-forming assay.