Figure 2

Overview of the neo -based retrotransposition assay in HeLa cells (adapted from[60]) and basal retrotransposition levels. (A) HeLa cells were transfected with plasmids encoding the neo-marked zebrafish L2-2 (ZfL2-2) [11], human L1 (hL1) (specifically L1.2) [60], zebrafish L2-1 (ZfL2-1) [11], or eel L2 (UnaL2) [12] retrotransposons. The neomycin resistance cassette (neo), inserted within the 3′ UTR region of each tested retrotransposon in the opposite direction, is interrupted by an intron from the human γ-globin gene in the sense orientation. Transcription of the retrotransposon DNA containing the cassette, splicing of the γ-globin intron, reverse transcription and integration in the genome are required for expression of the neo gene. The number of G418 resistant colonies obtained after 12 days of selection is proportional to the number of successful retrotransposition events. The position of primers used to discriminate between the intronless and unspliced retrotransposon copies and the sizes of PCR products are indicated. (B) Representative experimental results obtained in the absence of APOBEC proteins after selection of neomycin resistant colonies for 12 days. The approximate basal retrotransposition rates of each tested retrotransposon, expressed, in average number of colonies per plate, were: 4958 (split into 5 plates for counting) for hL1, 782 for ZfL2-2, 54 for ZfL2-1 and 101 for UnaL2 retroelements. pCMV, cytomegalovirus promoter; SVpA, SV40 poly A signal; f, neo437s primer; r, neo1808as primer. To rule out the possibility that the observed LINE inhibition was due to a non-specific toxicity of the tested AID/APOBEC proteins, the number of G418 resistant colonies obtained upon co-transfection of HeLa cells with an AID/APOBEC encoding and a neo-expressing pcDNA3.1 plasmid was determined (Additional file 1: Figure S1) as described in [53] and [61].