Characterization of L1 retrotransposition with high-throughput dual-luciferase assays.

Abstract:

:Recent studies employing genome-wide approaches have provided an unprecedented view of the scope of L1 activities on structural variations in the human genome, and further reinforced the role of L1s as one of the major driving forces behind human genome evolution. The rapid identification of novel L1 elements by these high-throughput approaches demands improved L1 functional assays. However, the existing assays use antibiotic selection markers or fluorescent proteins as reporters; neither is amenable to miniaturization. To increase assay sensitivity and throughput, we have developed a third generation assay by using dual-luciferase reporters, in which firefly luciferase is used as the retrotransposition indicator and Renilla luciferase is encoded on the same or separate plasmid for normalization. This novel assay is highly sensitive and has a broad dynamic range. Quantitative data with high signal-to-noise ratios can be obtained from 24- up to 96-well plates in 2-4 days after transfection. Using the dual-luciferase assays, we have characterized profiles of retrotransposition by various human and mouse L1 elements, and detailed the kinetics of L1 retrotransposition in cultured cells. Its high-throughput and short assay timeframe make it well suited for routine tests as well as large-scale screening efforts.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Xie Y,Rosser JM,Thompson TL,Boeke JD,An W

doi

10.1093/nar/gkq1076

subject

Has Abstract

pub_date

2011-02-01 00:00:00

pages

e16

issue

3

eissn

0305-1048

issn

1362-4962

pii

gkq1076

journal_volume

39

pub_type

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