Structure of the prototype vectors used for mutational analysis and experimental scheme of the transposition assay. (A) Vector structures containing the full-length type (top) and IΔ1 type (bottom) IAP element. To detect transposition, a GFP cassette is inserted in pFL and pDE1, in reverse orientation against the IAP element. The GFP gene is interrupted by an intron that is inversely oriented relative to the GFP gene. Solid and open arrows indicate orientation of the GFP gene and the intron, respectively. pFLex was used to provide full-length type autonomous IAP protein in trans to the cotransfected IAP element. The U3 region of 5′ LTR is replaced with the CAG promoter and the chimeric nature of the LTR is indicated by CAG/LTR. Pro, promoter; pA, polyadenylation signal. (B) Scheme of transposition assay. (Top) GFP is not expressed prior to transposition because of the inversely oriented intron within the GFP gene. (Middle) The intron is spliced out following transcription. GFP is not expressed from this transcript because the transcript is antisense relative to the GFP gene. (Bottom) The GFP expression unit is generated following reverse-transcription and integration into the genome. Evidence of a transposition event is detected by flow cytometry.
