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Nucleic Acids Research, 2000, Vol. 28, No. 6 1418-1423
© 2000 Oxford University Press

Determination of L1 retrotransposition kinetics in cultured cells

Eric M. Ostertag, Eline T. Luning Prak, Ralph J. DeBerardinis, John V. Moran1 and Haig H. Kazazian Jr*

Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA and 1Department of Human Genetics, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA

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, L1RP and L1.3. L1RP 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.

* To whom correspondence should be addressed. Tel: +1 215 898 3582; Fax: +1 215 573 7760; Email: kazazian@mail.med.upenn.edu


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