Generation of an inducible and optimized piggyBac transposon system.
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.
Cadiñanos
Juan
J
The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK.
Bradley
Allan
A
D000818:Animals; D002460:Cell Line; D003062:Codon; D004251:DNA Transposable Elements; D053595:Embryonic Stem Cells / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D023281:Genomics / Q000379:methods*; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D015202:Protein Engineering; D020079:Terminal Repeat Sequences; D019895:Transposases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D003062:Codon; D004251:DNA Transposable Elements; D019895:Transposases