Figure 2

Chicken DT40 cell culture-based assay for L1 retrotransposition. (A) Schematic of the human L1-eGFP vector. The L1 transcription is driven by a CMV promoter in addition to the L1 5'UTR. The human L1 retrotransposon contains an intron-interrupted EGFP reporter in the 3'UTR region with its own promoter and polyadenylation signal. The EGFP cassette is in antisense orientation relative to L1. Only when EGFP is transcribed from the L1 promoter, spliced, reverse-transcribed and integrated into the genome does a cell become GFP-positive. As a negative control, inactive L1 (pCMV-ΔRP99-eGFP) that contained two missense mutations in ORF1 that abolish retrotransposition was used. Arrows depict the location of the geno-5 (left) and geno-3 (right) primers used in the PCR assay shown in below. SD = splice donor; SA = splice acceptor. (B) Detection of L1 retrotransposition events in chicken cells. The geno-5 and geno-3 primers that flank the intron in GFP were used for PCR and analysed on a 1.2% agarose gel. PCR products of 1.49 kb (corresponding to the intron-containing transgene) and approximately 0.5 kb that lacks the 909 bp intron (corresponding to the transposed insertion) are shown. Negative, genomic DNA from wild-type DT40 cells; Vector, 5 ng plasmid DNA; Marker, 1 kb-plus DNA marker (Invitogen). (C) Quantitative RT-PCR analysis of the L1 transcript of human L1 elements in control (Dox-) and Dicer-deficient (Dox+) cells after addition of 2 μg/ml Dox for 72 h. Data are normalized to that of chicken β-actin transcripts. Error bars show s.d. (n = 6).