Fig. 1

Retroviral vectors used for expression of Fv1. a Schematic diagrams showing the plasmids for the previously described non-inducible bicistronic retroviral vectors, LxIG-Fv1 and LxIY-Fv1. After provirus formation, transcription of bicistronic mRNA is driven by the MLV U3 promoter, and translation of Fv1 is initiated at the 5′ CAP while the translation of the fluorescent protein is initiated from an EMCV IRES element. b Schematic diagrams showing the plasmids for the novel inducible bicistronic retroviral vectors used in this study. After integration into cells expressing the rtTA3 transactivator, the transcription level of bicistronic RNA driven by the inducible TRE3G promoter is dependent on the concentration of added doxycycline. The translation of the fluorescent protein is initiated at the 5′ CAP. For TGIx-Fv1 and TYIx-Fv1, the translation of Fv1 is initiated from an EMCV IRES. For TGx-Fv1 and TYx-Fv1, the Fv1 ORF is placed downstream to that of the fluorescent protein, and is most likely translated by a ribosome re-initiation mechanism. CMV, Cytomegalovirus immediate early promoter; R, repeated element; U5, unique 5′ element; U3, unique 3′ element; ΔU3, U3 region with deletion in enhancer sequence; Ψ, packaging signal; B1, Gateway cloning attB1 site; B2, Gateway cloning attB2 site; TRE3G, doxycycline-inducible TRE3G promoter. c FACS plots showing the separation of GFP+ and GFP− populations by FACS analysis in MDTF-R18 cells transduced with LxIG-Fv1^b, TGx-Fv1^b and TGIx-Fv1^b retroviral vectors. Cells transduced with the inducible vectors were treated with 10 μg/mL doxycycline for 24 h prior to analysis