Fig. 1

HsMar1 excision in two genetic backgrounds: human (HeLa cells) and non-human (CHO cells). a HsMar1 excision cassette before (top) and after (down) the expected HsMar1 excision. PiggyBac (PB) TIRs are represented as blue arrows ending the cassette; the HsMar1 recombinant copy is drawn in orange with its 5′ and 3’TIRs (orange arrows) and its transposase (Tpase) (orange rectangle); the LoxP recombination sites of CRE recombinase are in grey and GFP in green. The three promoters (pCMV, endogenous HsMar1 and that driving the puromycin resistance gene) are shown as thin arrows coloured in green, orange and grey respectively. b Excision assays exemplified for two cell lines (HeLa-D2 and CHO-A6). HeLa or CHO were transfected with HSMAR-RA (150 or 1050 ng), CRE (150 ng) expressing plasmid or an empty plasmid (pCS2). Transfection efficiency is controlled by transfecting a GFP expressing plasmid. 48 h after transfection, excision is controlled by GFP expression. c RT-PCR analyses of recombinant cell lines (for HeLa: B3, D2 and D4, left panel and for CHO: A6, A8 and B4, right panel) and their respective empty cell lines (no Tpase). BET-stained agarose gels are shown. Bands detected in HeLa cells are a mixed of PCR products obtained from both endogenous and recombinant HsMar1 copies. d Excision sites amplification in recombinant HeLa-D2 and CHO-A6 cell lines 48 h post- transfection; the different expressing plasmids use to promote excision are indicated above and amounts are as in (B) (pCS2: control plasmid). BET-stained agarose gels of PCR products are shown. The expected band is pinpointed (left margin)