Purpose: Long interspersed element type 1 (LINE-1, L1) occupies approximately 17% of human genome, and the majority of these L1s are inactive due to 5’ truncation and other mutations. Recently, targeted or whole genome sequencing has revealed abnormal L1 activities across multiple cancer types. Animal models are critical for investigating L1 insertion events and the associated consequences in vivo. Although specific mouse L1 loci have been reported and characterized, the overall L1 activity profile of the mouse genome is still unknown. There are at least 29 mouse-specific L1 families, eight of which are potentially capable of retrotransposition. In this project, we aim to investigate and compare promoter activities of different mouse L1 subfamilies. The rationale is that transcription is the initial and critical regulatory step for active retrotransposition. Meanwhile, The most active L1 element also can be served as transgene to generate a novel endogenous mouse model which can be served for drug screening in future studies.
Methods: A streamlined cloning method was established for screening the promoter activity. The dual-luciferase reporter system was utilized, in which the firefly luciferase signal indicates the promoter activity and the Renilla luciferase signal serves as a control for transfection efficiency (Fig.1).
Results: So far, we have cloned a large number of full-length L1 elements across all potential active L1 families from the mouse genome and subcloned promoters from those L1 loci. By high-throughput dual-luciferase assay, we are able to investigate L1 promoter activity and L1 retrotransposition activity across different cell lines (Fig. 2). In addition to evaluating the importance of promoter to retrotransposition activity, we will also investigate the sequence determinants of promoter activity among different mouse L1 families.
Conclusion: Our dual-luc reporter assay can be applied for promoters, gene expression in the manner of high-throughput. L1 promoters have subfamily specific activity profile and are highly dependent on biologically supportive environments.