Functional investigation of miRNAs in 32D cells. (A) Overview of the barcoded retroviral miRNA expression vector MSCV-BC-miRNA. MiRNA expression is driven by the viral LTR promoter. This vector contains the miRNA and ∼ 250 bp endogenous flanking sequences. To allow selection of infected cells, we incorporated a dual selection cassette that consist of the puromycin-N-acetyltransferase gene fused to a segment of the FMDV 2A peptide followed by the gene coding for GFP and a 24 BP barcode (BC) sequence. (B-C) Murine myeloid 32D cells were infected with MSCV-BC vectors containing different miRNAs or no miRNA as control (A1, EV) and sorted for GFP expression by flow cytometry. Equal number of cells were mixed and switched from IL-3- to CSF3-containing medium. Cell samples were taken at indicated time points and genomic DNA was isolated. The abundance of the different BC sequences was measured with the Luminex technology. The ratios of the barcode A8 (miR-292) and barcode F5 (miR-93) signals to the A1 barcode (EV) signal of a representative result (of 2 experiments for miR-93 and 3 experiments for miR-292) are shown. (D) Micrographs showing morphology of control 32D cells (A1-EV), 32D-A8-miR-292 and 32D-F5-miR-93 cells on day 7 of CSF3 treatment. Bar indicates 10 μm.