Fig. 2

L1 retrotransposition rate is not significantly different in CSA-deficient cells (CSA-) and in the stably complemented CSA-deficient cells (CSA+). a Schematic of L1 retrotransposition assay. The L1.3 element tagged at the 3′ end with the mneo retrotransposition sensor is inserted in a pCEP4 vector (JM102/L1.3 vector). The retrotransposition cassette consists of a neomycin resistance (NeoR) gene in antisense orientation relative to the L1 element and expressed from its own promoter. The NeoR gene is not functional in the retrotransposition cassette because it is interrupted by an intron in L1 sense orientation. The NeoR gene becomes functional only after transcription, splicing, reverse transcription of L1 mRNA and insertion of L1 cDNA. b Schematic of the timing of the L1 retrotransposition assay. CSA- and CSA+ cells are seeded the day before transfection with JM102/L1.3 (L1.3-mneo-WT) or JM102/D702A/L1.3 (L1.3-mneo-RT(−)) expression vector. Two days after transfection, G418 selection is added to the growth medium and cells are kept under selection for 14 days. At the end of the assay, cells are fixed and stained and the number of NeoR colonies is determined. c CSA-deficient cells (CSA-) and the stably complemented version (CSA+) were transfected with JM102/L1.3 (L1-mneo) or JM102/D702A/L1.3 (L1-mneo-RT(−)) construct. Colony formation was assayed after two weeks under neomycin selection. The graph shows the relative colony number (average ± S.D.) of three independent experiments. Values are normalized to L1.3 WT vector. No significant differences (p > 0.05, two-tailed two sample Student’s T-test) were observed between the L1-mneo expression constructs in the different CSA+ and CSA- cells. Representative examples of NeoR colony formation from L1 retrotransposition assay in CSA+ and CSA- cells were presented below the graph. No colonies were detected with the L1 element with a defective RT. d CSA-deficient cells (CSA-) and the complemented version (CSA+) were co-transfected with TAM102/L1.3 (L1 mblast), or TAM102/H230A/L1.3 (L1 (en-)-mblast) construct and pIRES2-EGFP vector, a vector carrying a constitutive NeoR expression cassette. Colony formation due to random integration of this transfected plasmid was assayed after two weeks under neomycin selection. The L1 expression constructs were only included as a functional L1 and a defective (en-) L1 so that the experiment can simultaneously test for differences in the CSA- and CSA+ cells for transfection, colony formation and potential toxicity from the L1. The graph shows the relative colony number (average ± S.D.) of three independent experiments. Values are normalized to L1.3 WT vector. No significant differences (p > 0.05, two-tailed two sample Student’s T-test) were observed between the different L1 expression constructs in the different cell lines. Representative examples of NeoR colony formation from this L1 toxicity assay in CSA+ and CSA- cells are presented below the graph