Characterization of L1 retrotransposition with high-throughput dual-luciferase assays.
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.
School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, WA 99164, USA.
Yi
Y
Xie
James M
JM
Rosser
Tina L
TL
Thompson
Jef D
JD
Boeke
Wenfeng
W
An
D000818:Animals; D017930:Genes, Reporter; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D007700:Kinetics; D020084:Long Interspersed Nucleotide Elements; D049409:Luciferases, Firefly / Q000032:analysis / Q000235:genetics*; D049410:Luciferases, Renilla / Q000032:analysis / Q000235:genetics*; D049408:Luminescent Agents; D051379:Mice; D008904:Miniaturization; D011401:Promoter Regions, Genetic; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology
D049408:Luminescent Agents; D018894:Reverse Transcriptase Inhibitors; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly