Pcl eco packaging vector

These assays were conducted as described previously (Jiang et al., 2012 (link); Li et al., 2012 (link)) with some modifications. Briefly, retroviruses or lentiviruses were produced in 293T cells by co-transfection of individual expression construct with the pCL-Eco packaging vector (IMGENEX, San Diego, CA) or the pMD2.G:pMDLg/pRRE:pRSV-Rev packaging mix (individually purchased from Addgene), respectively. The virus particles were harvested at 48 and 72 hours after transfection and added to cells with or without concentrating, and two rounds of “spinoculation” were performed to allow the infection of viruses.

MONOMAC-6 cells were maintained in RPMI 1640 supplemented with 10% FBS, 1% HEPES, 2 mM L-Glutamine, 100×Non-Essential Amino Acid (Invitrogen), 1 mM sodium pyruvate, 9 μg/ml insulin (Invitrogen) and 1% penicillin-streptomycin. Plasmids were transfected into MONOMAC-6 cells with Cell Line Nucleofector Kit V following program T-027 using the Amaxa® Nucleofector® Technology (Amaxa Biosystems, Berlin, Germany). Experiments were performed 48 hours after transfection.
The MLL-ENL-ERtm cell line was kept in RPMI 1640 supplemented with interleukin 3 (IL-3), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF), 10 ng/ml; SCF 100 ng/ml; 10% FBS and 1% penicillin-streptomycin. 4-Hydroxy-tamoxifen (4-OHT) (Sigma-Aldrich, St. Louis, MO) was added at a 100 nM final concentration as a 1 mM stock solution in ethanol.
Retrovirus was used to transduce MLL-ENL-ERtm cells. Retrovirus for each construct was produced in 293T cells by co-transfecting the retroviral construct and pCL-Eco packaging vector (IMGENEX, San Diego, CA) as previously described[52 (link); 53 (link)]. Rat1a cells were used to determine the viral titer. BM progenitor Cells were co-transduced with MSCV-neo vector or MSCV-neo-MLL-AF9 together with MSCV-PIG vector or MSCV-PIG-miR-26a, or MSCV-PIG-miR-29a constructs, respectively, through “spinoculation”.