Figure 7. Synthetic L1/ORFeus transgene and progeny retrotransposon insertions.

(a) The transgene construct or donor element consists of the following sequence elements from 5’ to 3’: (i) a composite CMV IE enhancer/modified chicken β-actin promoter, designated “CAG”. (ii) synthetic L1 ORF1, ORF2 and 5’ portion of 3’UTR. (iii) Herpes simplex virus thymidine kinase poly(A) signal (boxed inverted letter A) in antisense orientation to polyadenylate gfp mRNA. (iv) gfp (green block arrow), a modified version of EGFP coding sequence. The gfp ORF is in antisense orientation relative to L1 and interrupted by intron 2 of human β-globin gene, which is in sense orientation relative to L1; gfp serves as a “retrotransposition indicator gene”. (v) Rous sarcoma virus LTR promoter in antisense orientation relative to L1, which drives gfp transcription (boxed inverted P for promoter). (vi) β-globin poly(A) signal (boxed upright letter A). Numbered arrows above the diagram represent locations of genotyping PCR primers. Region used to generate Southern blotting probes is indicated (purple line). (b) Structure of a representative progeny element. A typical progeny insertion is 5’ truncated, intronless, ends in a poly(A) tail (AAA) and is flanked by target site duplications (gray triangles) and target genomic DNA sequences (wavy solid lines). Primers 1 and 1’ (intron flanking primers) amplify a longer product when derived from the donor element (A) than from the progeny insertions (B); product length differs by the length of the intron. Primers 2 and 1’ (primer 2 is the “intron spanning” primer that spans the splice junction give rise to a product only from progeny retrotransposition events. Primers 3 and 3’ are control primers that give rise to products of constant length for donor and progeny elements.