Figure 2

The time course of L1 retrotransposition in HeLa Tet-ORFeus cells. (A) Fluc and Rluc activities from cell lysates. Cells were seeded in 96-well plates (for luminescence) or 60 mm dishes (for protein and gDNA analyses) in the absence of doxycycline and harvested at the indicated time points. Error bars represent mean±SE (n = 6). All readings were compared with the 0 h control (**P <0.01). (B) Time-dependent increase of ORF1p expression. ORF1p and β-actin were detected by western blot. Murine embryonal carcinoma cells (F9) were used as a positive control for ORF1p. The parental HeLa-tTA cells and uninduced HeLa Tet-ORFeus cells were used as negative controls. (C) Confirmation of L1 retrotransposition by end-point PCR. Genomic DNA was amplified by an intron-flanking primer pair. The presence of a band of 250 bp is diagnostic for intron removal; the intron-containing donor DNA is amplified as a band of 1150 bp. NTC, no template control. Dox+, gDNA from cells cultured in the presence of doxycycline for 48 h. Fluc plasmids with or without the intron were used as controls. Molecular weight was indicated by the 1 kb Plus DNA Ladder (Invitrogen). (D) Quantification of L1 insertions by qPCR. The number of L1 insertions in gDNA was determined by a TaqMan-based qPCR assay. qPCR signals were normalized by setting signals from the 48 h time point to 1. The normalized signals from each time point were then compared with the 0 h time point by two-tailed Student’s t-test. P values are indicated (**P <0.01). Error bars represent mean±SE (n = 3).