Fig. 2From: L1 retrotransposition in the soma: a field jumping aheadInterpreting results from the engineered L1-EGFP reporter assay. a. The L1-EGFP reporter gene [123] comprises a full-length human or mouse L1 (e.g. [41, 122, 291]) tagged with a cassette incorporating EGFP and its promoter in the opposite orientation to the L1, followed by an SV40 polyA signal. Transcription of the combined L1-EGFP reporter, followed by splicing (dotted line) of an intron in the EGFP gene, prepares the L1-EGFP mRNA for reverse transcription and integration into the genome via target-primed reverse transcription (TPRT). The L1-EGFP reporter has been introduced in vitro as a plasmid [19–21, 126, 171] and also as a rodent transgene [8, 9, 21, 27, 116]. b. Successful TPRT-mediated retrotransposition of the engineered L1 mRNA yields an intact EGFP gene, leading to GFP+ cells (true positives). c. Mobilization of the engineered L1 mRNA may occur through TPRT but, due to severe 5′ truncation removing the L1 entirely, or 5′ inversion/deletion [95, 292] the EGFP gene may be incompetent at its 3′ end, and therefore retrotransposition results in GFP− cells (false negatives). d. The engineered L1 mRNA may be retrotransposed, yielding a functional EGFP gene, but the EGFP promoter is epigenetically silenced [126], leading to GFP− cells (false negatives). PCR-based assays targeting the EGFP splice junction can, however, identify instances where successful retrotransposition is not marked by EGFP expression [19, 46, 123, 126]. e. Finally, retrotransposition of the engineered L1 mRNA may simply have not occurred in GFP− cells (true negatives)Back to article page