LINE-1 Cultured Cell Retrotransposition Assay

Abstract

The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells.

Keywords: Alu; LINE-1; Mammalian cultured cells; Retrotransposition assay; trans-complementation assay.

Figure 1. LINE-1 Retrotransposition Assay

(A) A full-length retrotransposition-competent LINE-1 contains the mneoI reporter cassette (orange box) in the 3’UTR in the opposite orientation with respect to LINE-1 (pJM101/L.13) transcription. The reporter gene, neomycin phosphotransferase (backwards NEO) is interrupted by an intron (hatched box), which is in the same orientation with respect to LINE-1 transcription. The reporter cassette has its own promoter (upside down black arrow) and polyadenylation signal (upside down small black lollipop). The pCEP4 plasmid backbone encodes for the EBNA-1 (EBNA-1) viral protein and contains an origin of viral replication (oriP) and hygromycin B-resistance gene (HYG^R) for plasmid replication and hygromycin-selection, respectively, in mammalian cultured cells. The backbone also has a bacterial origin of replication (ori) and an ampicillin-resistance gene (AMP^R) for replication and ampicillin-selection, respectively, in E.coli. The LINE-1 is transcribed from the CMV promoter (black triangle labeled “CMV”) or its promoter in the 5’UTR (gray arrow) and transcription is terminated at an SV40 polyadenylation signal (large black lollipop). Once transcribed, L1 ORF1p (yellow circles) and ORF2p (blue circle) are translated from the L1 mRNA (grey, yellow, blue, and orange line followed by “AAAA_n”) and the intron is spliced out. Only upon reverse transcription and integration into a genomic locus (5’ truncated blue and orange box), which is flanked by target-site duplications (TSD, red wavy lines), can the NEO gene be expressed to confer drug-resistance. (B) A timeline of the assay is depicted and described in the Methods. Days of the protocol are noted above and the corresponding days post-transfection (d0–14) are noted below. The end result of the assay is depicted under d14. The pJJ101/L1.3 construct is a wild type LINE-1 with a blasticidin deaminase (mblastI) reporter cassette and its retrotransposition results in many drug-resistant colonies. The pJJ105/L1.3 construct contains a LINE-1 reverse transcriptase mutant, cannot complete retrotransposition, and does not result in any drug-resistant colonies.

Figure 2. Alu Retrotransposition (trans-complementation) Assay

Retrotransposition of Alu requires the LINE-1 ORF2 protein (43). For the Alu trans-complementation assay, the reporter plasmid contains the Alu sequence (light blue box) and is tagged with a modified NEO retrotransposition indicator cassette (neo^Tet) interrupted by a self-splicing group I intron (checkered box) (81). The neo^Tet cassette is followed by a variably sized poly A tail (A_n) and the RNA pol III terminator sequence (red lollipop). The Alu plasmid must be co-transfected with a plasmid (pCEP5’UTR-ORF2pΔneo) expressing LINE-1 ORF2p (blue circle) to detect retrotransposition in HeLa cells. The resulting G418-resistant colonies generally contain de novo full-length Alu retrotransposition events flanked by target-site duplications (TSD, red wavy lines).

Figure 3. Co-transfection of LINE-1 with cDNAs

The LINE-1 retrotransposition assay is carried out as described in Figure 1 except that a plasmid expressing pK_A3A (purple box) is co-transfected with a LINE-1 expression construct (pJM101/L1.3, see Figure 1A for details). In parallel, pK_A3A is co-transfected with a reporter control, pU6iNEO, which expresses the neomycin phosphotransferase gene without the requirement for retrotransposition (i.e., the neomycin phosphotransferase gene lacks an intron). Both sets of co-transfected cells are grown under drug-selection and the resulting drug-resistant colonies (dark purple circles) are counted. The parallel pU6iNEO experiment is essential to determine to what extent the effects of A3A overexpression are LINE-1 specific. Note the greater decrease in G418-resistant colonies in “LINE-1 + A3A” than “pU6iNEO + A3A” (79).