Figure 2. Enrichment of the L1 RNP.

(A) A timeline of the LEAP 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. (B) The pJM101/L1.3 L1 construct contains the L1.3 element (accession no. L19088). The pCEP4 plasmid (Life Technologies) backbone encodes for the EBNA-1 (EBNA-1) viral protein and contains an origin of viral replication (oriP), a hygromycin B-resistance gene (HYG^R), the cytomegalovirus (CMV) promoter (large black triangle) and an SV40 polyadenylation signal (large black lollipop) for plasmid replication, hygromycin-selection, and transcription, respectively, in mammalian cultured cells. The pCEP4 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. (For details of the mneoI reporter cassette, please see the “LINE-1 Cultured Cell Retrotransposition Assay” chapter in this volume.) After transfection and hygromycin B-selection in HeLa-JVM cells, the cells are lysed and subjected to high-speed centrifugation through a sucrose cushion. After centrifugation, cellular RNPs are enriched in the pellet fraction. This fraction contains the L1 RNA bound by L1 ORF1p and ORF2p, which minimally constitutes the L1 RNP (see Figure 1). (C) The pES2TE1 L1 construct (18), like pJM101/L1.3, contains the L1.3 element in a pCEP4 (Life Technologies) backbone. Unlike pJM101/L1.3, the pES2TE1 construct encodes a T7-tagged (orange diamond) ORF1p and a FLAG-HA-tagged (purple diamond) ORF2p (18). After transfection and hygromycin B selection, HeLa cells are lysed and subjected to immunoprecipitation using an anti-FLAG antibody conjugated to beads (red circle with black “Y”). Immunoprecipitated complexes contain L1 ORF1p and ORF2p bound to its encoding RNA, which minimally constitutes the L1 RNP.