Figure 4: De novofull-length L1 insertions retain retrotransposition competencyin vitro.

Intact, full-length L1 insertions L1-dn6-2.2 and L1-dn6-5.4 were obtained from two independent genomic PCR reactions amplifying the L1-dn6 de novo insertion, tagged with an mblastI retrotransposition indicator cassette, and inserted into an episomal expression plasmid where they were transcriptionally controlled by the CMV promoter. Resulting L1 reporter plasmids pJJ101/L1-dn6-2.2 and pJJ101/L1-dn6-5.4 were submitted to the L1 retrotransposition reporter assay (see Methods). HeLa cells were transfected with the L1-dn6 reporter plasmids or with positive and negative control L1 reporter plasmids pJJ101/L1.3 and pJJ101/L1.3-D702A, respectively. Blastidicin-S resistant cells arise only if engineered L1 retrotransposition has occurred. pJJ101/L1.3 was used for normalization (100% activity). pJJ101/L1.3-D702A contains a single point mutation in the L1 reverse transcriptase domain. The bar diagram depicts arithmetic mean±s.d. of three independent retrotransposition reporter assays of the engineered L1-dn6 elements relative to L1.3. Black hexagon, SV40 polyadenylation signal; grey arrows, TSDs flanking a 5′-truncated de novo L1 insertion. Blast(s), Blastidicin-S sensitive; Blast(r), Blastidicin-S resistant; SD, splice donor; SA, splice acceptor.