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Generation of an inducible and optimized piggyBac transposon system.

Published version
Peer-reviewed

Type

Article

Change log

Authors

Cadiñanos, Juan 

Abstract

Genomic studies in the mouse have been slowed by the lack of transposon-mediated mutagenesis. However, since the resurrection of Sleeping Beauty (SB), the possibility of performing forward genetics in mice has been reinforced. Recently, piggyBac (PB), a functional transposon from insects, was also described to work in mammals. As the activity of PB is higher than that of SB11 and SB12, two hyperactive SB transposases, we have characterized and improved the PB system in mouse ES cells. We have generated a mouse codon-optimized version of the PB transposase coding sequence (CDS) which provides transposition levels greater than the original. We have also found that the promoter sequence predicted in the 5'-terminal repeat of the PB transposon is active in the mammalian context. Finally, we have engineered inducible versions of the optimized piggyBac transposase fused with ERT2. One of them, when induced, provides higher levels of transposition than the native piggyBac CDS, whereas in the absence of induction its activity is indistinguishable from background. We expect that these tools, adaptable to perform mouse-germline mutagenesis, will facilitate the identification of genes involved in pathological and physiological processes, such as cancer or ES cell differentiation.

Description

Keywords

Animals, Cell Line, Codon, DNA Transposable Elements, Embryonic Stem Cells, Gene Expression Regulation, Enzymologic, Genomics, Humans, Mice, Mutagenesis, Protein Engineering, Terminal Repeat Sequences, Transposases

Journal Title

Nucleic Acids Res

Conference Name

Journal ISSN

0305-1048
1362-4962

Volume Title

35

Publisher

Oxford University Press (OUP)