Determination of L1 retrotransposition kinetics in cultured cells
Open accessAbstract
L1 retrotransposons are autonomous retroelements that are active in the human and mouse genomes. Previously, we developed a cultured cell assay that uses a neomycin phosphotransferase ( neo ) retrotransposition cassette to determine relative retrotransposition frequencies among various L1 elements. Here, we describe a new retrotransposition assay that uses an enhanced green fluorescent protein (EGFP) retrotransposition cassette to determine retrotransposition kinetics in cultured cells. We show that retrotransposition is not detected in cultured cells during the first 48 h post-transfection, but then proceeds at a continuous high rate for at least 16 days. We also determine the relative retrotransposition rates of two similar human L1 retrotransposons, L1(RP)and L1.3. L1(RP)retrotransposed in the EGFP assay at a rate of approximately 0.5% of transfected cells/day, approximately 3-fold higher than the rate measured for L1.3. We conclude that the new assay detects near real time retrotransposition in a single cell and is sufficiently sensitive to differentiate retrotransposition rates among similar L1 elements. The EGFP assay exhibits improved speed and accuracy compared to the previous assay when used to determine relative retrotransposition frequencies. Furthermore, the EGFP cassette has an expanded range of experimental applications.
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The episomal L1-EGFP reporter plasmid and the L1 5′UTR-Luciferase reporter plasmid used in this study were both described previously.
“The episomal L1-EGFP reporter plasmid and the L1 5′UTR-Luciferase reporter plasmid used in this study were both described previously25,50.”
From SIRT6 represses LINE1 retrotransposons by ribosylating KAP1 but this repression fails with stress and age (2014) · cites this paper as [25]
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- Authors
- Ostertag, E. M.
- Affiliations
- University of Pennsylvania (US)
- Corresponding author
- Ostertag, E. M.
- ORCID
- Ostertag, E. M.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2000-03-15
- Volume
- 28
- Issue
- 6
- Pages
- 1418-1423
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC111040
- OpenAlex
- W2150649207
- Cited by
- 291
- References
- 29
- Citations by year
- 2026: 8 · 2025: 16 · 2024: 10 · 2023: 9 · 2022: 16 · 2021: 12 · 2020: 15 · 2019: 17 · 2018: 14 · 2017: 21 · 2016: 11 · 2015: 9 · 2014: 7 · 2013: 17 · 2012: 9
- FWCI
- 8.97
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Nucleic Acids Research (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- University of Pennsylvania
- National Institutes of Health
- Topics
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Chromosomal and Genetic Variations
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomic variations and chromosomal abnormalities
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genome Rearrangement Algorithms
- Keywords
- LINE-1 retrotransposons, cultured cells, transposable elements
- MeSH terms
- Cinnamates, Drug Resistance, False Positive Reactions, Globins, HeLa Cells, Humans, Hygromycin B, Introns, Kinetics, Luminescent Proteins, Microscopy, Fluorescence, Puromycin, Recombination, Genetic, RNA Splicing, Sensitivity and Specificity, Time Factors, Transfection, Mutagenesis, Insertional, Genes, Reporter, Retroelements, 3' Untranslated Regions, Green Fluorescent Proteins
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