Experimental strategy for assaying RC-L1 expression and retrotransposition. (A) L1 element consists of the 5'- and 3'-UTRs and two ORFs. The EGFP retrotransposition cassette is cloned into the L1 3'-UTR in the antisense orientation. L1 elements tagged with the EGFP cassette are cloned into the pCEP4-based mammalian expression vector with puromycin resistance gene. RC-L1-EGFP-expressing cells are sorted by FACS and retrotransposition is detected by EGFP fluorescence using fluorescence microscopy. (B) Confirmation of L1 retrotransposition by PCR as revealed by a 342 bp product. MCF-7 cells-expressing L1-EGFP. Serial passaged cells (P1–P4) show the spliced form of EGFP at 342 bp. No DNA template used as negative control. L1-EGFP plasmid showing the unspliced form at 1243 bp used as a positive control. 1 Kb ladder used as a molecular weight marker.