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Fig. 3 | Mobile DNA

Fig. 3

From: Restricting retrotransposons: a review

Fig. 3

Cell culture retrotransposition assay reporter gene cassettes come in a variety of flavors. a. LINE-1 assays. A retrotransposition-competent L1 and reporter cassette is cloned in pCEP4 (Invitrogen)-based vectors, which encode EBNA-1 and OriP and so replicate in primate cells. Variants of the vectors also contain or lack an exogenous promoter upstream of the L1, and encode resistance to hygromycin or puromycin permitting antibiotic selection of transfected cells. mneoI and mblastI reporter cassettes confer drug resistance to cells having a retrotransposition event. These cells are expanded in culture to form colonies, fixed, stained, and the number of colonies scored. The mEGFPI cassette fluorescently marks cells with retrotransposon insertions and allows their numbers to be counted by flow cytomentry. Firefly luciferase gene mFlucI reporter vectors may be cotranfected with pGL4.73 (Promega) or other vector which constitutively expresses renilla luciferase from transfected cells. Following cell lysis, retrotransposition levels, indicated by firefly luciferase, are adjusted to renilla expression to control for differences in transfection efficiency. The mGlucI cassette expresses Gaussia luciferase which when secreted into the media serves as an effective read-out of accumulated retrotransposition events. Levels of Gluc may be normalized to those of Cypridina luciferase (which is also secreted and does not cross-react with Gluc) constiitutively expressed from the cotransfected pSV40-CLuc vector (NEB). Simply by sampling small aliquots of cell culture media, retrotransposition may be assessed in a single well at multiple time points without cell lysis. Luciferase-based reporter cassettes are amenable to HT retrotransposition screening. b. The Alu assay. An active Ya5 Alu and neoTET cassette interrupted by a Tetrahymena thermophila self-splicing 23S rRNA Group I intron is cloned between the 7SL pol III enhancer and terminator. When this construct is co-expressed with L1 ORF2 alone or a full-length retrotransposition-competent L1, Alu RNAs are reverse transcribed along with the spliced npt gene and integrated into the genome to confer neomycin resistance. Abbreviations: 7SL enh, 7SL enhancer; 7SL TTTT, 7SL transcription terminator; ampR, ampicillin resistance gene; bsd, blasticidin S deaminase gene; CMV, cytomegalovirus promoter; EBNA-1, Epstein-Barr nuclear antigen 1; EGFP, enhanced green fluorescent protein; L mon, left monomer; mini, chimeric mini-intron of the plasmid psiCHECK-2 (Promega); npt, neomycin phosphotransferase gene; oriP, latent origin of replication; pCI: synthetic intron from pCI (Promega); R mon, right monomer; SA, splice acceptor; SD, splice donor; SV40, simian virus 40 early enhancer/promoter; TET, T. thermophila self-splicing intron; TK, herpes simplex virus thymidine kinase poly(A) signal

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