Gene-trap tTA vector design and in vitro testing A). The T2/GT2/tTA and T2/GT3/tTA vectors. The 'GT2' version is capable of mutating genes in one orientation while the 'GT3' version can mutate genes in both orientations upon insertion into a gene. (B- BamHI sites ) B) Compared to normal expression (left), when the T2/GT2/tTA SB transposon-based gene-trap vector inserts into a gene in the direction of transcription (right), endogenous splicing incorporates the IRES and tTA sequences and the bicistronic mRNA is prematurely truncated at the SV40 late polyadenylation site. The bicistronic mRNA allows cap-independent translation of the tTA molecule in addition to any peptide encoded by upstream exons. C) A stable, TRE-regulated luciferase cell line was created to test the T2/GT2/tTA/SVNeo transposon vector. After co-transfection with a plasmid source of transposase, G418R clones were individually expanded for analysis. D) Individual luciferase expression levels of G418R clone cell extracts from twelve control (pT2/SVNeo) and fifty gene-trap tTA clones from C. E) Incubation of three clones from C in media supplemented with 2 mM doxycycline results in 10- to 100-fold reduction of luciferase expression.