Long interspersed nuclear elements (LINEs/L1) are mobile genetic elements that undergo a process called retrotransposition. This process is replicative, and results in a new genomic L1 insertion originating from a pre-existing L1 element. Over millions of years of retrotransposition and new L1 elements being integrated into mammalian genomes, these L1s now make up a significant amount of human (17%) and mouse (18%) genomic DNA.
Previously developed retrotransposition assays allows us to visualize new L1 insertions using fluorescent protein reporters in cultured cells and in transgenic mouse models. However, these reporters may undergo epigenetic silencing, as has been observed in the human embryonal carcinoma cell line PA-1. In transgenic animal models, such silencing may result in an underestimate of L1 retrotransposition rates in vivo.
We have developed the L1-Cre system, which uses Cre recombinase, and a secondary conditional fluorescent reporter placed in a safe harbour locus to attempt to circumvent this reporter silencing in the hopes that it will allow for a more accurate representation of retrotransposition. Firstly, the L1-Cre system was compared to a retrotransposition reporter assay where retrotransposition directly delivers a fluorescent reporter gene, and it was found that both assays resulted in similar levels of mCherry positive cells. The L1-Cre system was then tested in the PA-1 cell line to determine if the system was successful in circumventing the observed reporter silencing. However, this model still required some adjustment.
Once optimized, L1-Cre in conjunction with R26-Confetti and Brainbow transgenic mice will be utilized to observe retrotransposition events more accurately. Depending on the outcomes of the Cre recombination, new retrotransposition events in these animal models will be tagged with different fluorescent markers, allowing us to track retrotransposition and better observe L1 mosaicism in vivo.