Structures of L1 element and L1-mless transgene. (A) Human full-length L1 contains a 5′UTR, ORF1, ORF2, 3′UTR, and a poly(A) tail. (B) L1LRE3 is tagged in its 3′UTR with a markerless retrotransposition cassette (mless) and is driven by the mouse Hsp70-2 promoter. Mless contains the human γ-globin intron in the same orientation as L1 (SD and SA denote splice donor and acceptor sites, respectively), flanked by splice acceptors from the human Bcl-2 gene (not shown). After retrotransposition, the intron is spliced, allowing de novo insertions to be distinguished from the original transgene by PCR with oligos L16045(30) and mlessfor2 (black arrows). The expected band pattern from this “genotyping PCR” is shown on the right. Gray arrows SP1–SP4 indicate insertion-specific oligos used in TAIL–PCR.
