Design of retroviral vectors and overexpression of cyclin D2 in hematopoietic progenitors. (A) The cyclin D2 gene was inserted in front of IRES and GFP into a retroviral construct.26 Cyclin D2 gene expression was driven from a MSCV promoter. The control vector contained GFP-IRES-L22Y. L22Y represents a human dihydrofolate reductase variant containing a leucine to tyrosine substitution in codon 22 of the gene.27 L22Y confers trimetrexate resistance; however, only GFP expression was used as a marker gene in this study. (B) LSK cells transduced with control (left) or cyclin D2 (right) containing vectors were cultured for an additional 3 days after transduction and analyzed by FACS for expression of cyclin D2 and GFP. Inserted in the upper left of the FACS plots are the control stainings for cyclin D2 (goat-antimouse-PE). Note that GFP⁺ and GFP^– control-transduced cells exhibited comparable levels of endogenous cyclin D2 protein. Results are from 1 of 3 experiments with similar results.