Nucleic Acids Research Advance Access published online on June 18, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm446
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Methods Online |
Generation of an inducible and optimized piggyBac transposon system
The Wellcome Trust Sanger Institute. Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK
*To whom correspondence should be addressed. Tel: +44-1223-494884; Fax: +44-1223-494714; Email: abradley{at}sanger.ac.uk
Received March 22, 2007. Revised May 14, 2007. Accepted May 18, 2007.
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.
GenBank accession numbers: EF587698 and EF587699.
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