Knockout Rat - LINE1 (L1) Retrotransposons

LINE1 (L1) Retrotransposons

Transposons and retrotransposons are valuable tools for unbiased gene discovery as mobile pieces of DNA used for gene disruption. Retrotransposons, such as LINEs (long interspersed nuclear elements), mobilize via a “copy and paste” mechanism and are abundant in many eukaryotic species. Several L1 retrotransposons have remained active in mice and humans. L1s contain a small internal promoter within a 5’ untranslated region to drive expression, two open reading frames (ORFs), and a 3’ untranslated region containing sequences for polyadenylation. The two ORFs encode proteins necessary for autonomous retrotransposition; ORF1 encodes an RNA-binding protein while ORF2 encodes a protein containing endonuclease (EN) and reverse transcriptase (RT) activity, which nick a site in DNA, then produce a copy via RT. These proteins exhibit an overwhelming specificity for binding to and acting on the transcript that encodes them, enabling near exclusive mobilization of the parental L1 RNA. Using the RT activity of the ORF2 protein, the transcribed L1 RNA is copied into DNA by a process termed target primed reverse transcription (TPRT), and integrated into the genome. Integration occurs with little bias for any particular genomic region, requiring a simple consensus sequence, 5’TTTT’A-3’ (along with minor variations of this sequence). Integrated L1 sequences are often truncated at the 5’ end, with an average total size of 1 Kb, many containing only 3’ terminal sequences.

The nature of retrotransposition endows the L1 with some unique advantages; L1 retrotransposons have an essentially unlimited supply of the insertional mutagen since it is continually transcribed from a promoter, which would be useful for applications where large numbers of mutations are needed in a single cell. L1 elements also demonstrate widespread genomic coverage, with a largely random distribution of insertions. L1 insertions at genomic sites are also irreversible, and thus any mutagenic event caused by an L1 insertion is “tagged” by L1 sequences.