Determination of L1 retrotransposition kinetics in cultured cells

Eric M. Ostertag, Eline T. Luning Prak, Ralph J. DeBerardinis, John V. Moran, Haig H. Kazazian

Research output: Contribution to journalArticle

178 Citations (Scopus)

Abstract

L1 retrotransposons am 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 Fates of two similar human L1 retrotransposons, L1(RP) and L1.3. L1(RP) retrotransposed in the EGFP assay at a rate.of ~0.5% of transfected cells/day, ~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.

Original languageEnglish (US)
Pages (from-to)1418-1423
Number of pages6
JournalNucleic Acids Research
Volume28
Issue number6
StatePublished - Mar 15 2000

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Retroelements
Cultured Cells
Long Interspersed Nucleotide Elements
Kanamycin Kinase
Human Genome
Transfection
enhanced green fluorescent protein

ASJC Scopus subject areas

  • Genetics

Cite this

Ostertag, E. M., Luning Prak, E. T., DeBerardinis, R. J., Moran, J. V., & Kazazian, H. H. (2000). Determination of L1 retrotransposition kinetics in cultured cells. Nucleic Acids Research, 28(6), 1418-1423.

Determination of L1 retrotransposition kinetics in cultured cells. / Ostertag, Eric M.; Luning Prak, Eline T.; DeBerardinis, Ralph J.; Moran, John V.; Kazazian, Haig H.

In: Nucleic Acids Research, Vol. 28, No. 6, 15.03.2000, p. 1418-1423.

Research output: Contribution to journalArticle

Ostertag, EM, Luning Prak, ET, DeBerardinis, RJ, Moran, JV & Kazazian, HH 2000, 'Determination of L1 retrotransposition kinetics in cultured cells', Nucleic Acids Research, vol. 28, no. 6, pp. 1418-1423.
Ostertag EM, Luning Prak ET, DeBerardinis RJ, Moran JV, Kazazian HH. Determination of L1 retrotransposition kinetics in cultured cells. Nucleic Acids Research. 2000 Mar 15;28(6):1418-1423.
Ostertag, Eric M. ; Luning Prak, Eline T. ; DeBerardinis, Ralph J. ; Moran, John V. ; Kazazian, Haig H. / Determination of L1 retrotransposition kinetics in cultured cells. In: Nucleic Acids Research. 2000 ; Vol. 28, No. 6. pp. 1418-1423.
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