Chromosomal transposition of PiggyBac in mouse embryonic stem cells

Abstract

Transposon systems are widely used for generating mutations in various model organisms. PiggyBac (PB) has recently been shown to transpose efficiently in the mouse germ line and other mammalian cell lines. To facilitate PB's application in mammalian genetics, we characterized the properties of the PB transposon in mouse embryonic stem (ES) cells. We first measured the transposition efficiencies of PB transposon in mouse embryonic stem cells. We next constructed a PB/SB hybrid transposon to compare PB and Sleeping Beauty (SB) transposon systems and demonstrated that PB transposition was inhibited by DNA methylation. The excision and reintegration rates of a single PB from two independent genomic loci were measured and its ability to mutate genes with gene trap cassettes was tested. We examined PB's integration site distribution in the mouse genome and found that PB transposition exhibited local hopping. The comprehensive information from this study should facilitate further exploration of the potential of PB and SB DNA transposons in mammalian genetics. ES cell ͉ Sleeping Beauty ͉ transposon DNA transposons are genetic elements that can relocate between genomic sites by a ''cut and paste'' mechanism. Since the discovery of the first DNA transposon in maize by Barbara McClintock (1), these elements have been extensively used for genetics and functional genomics in different organisms (2-7).Sleeping Beauty was the first DNA transposon system shown to be functional in mammalian cells. It has been tested for insertional mutagenesis in the mouse and rat germ lines (8-13), but its relatively low transposition efficiency and strong ''local hopping'' tendency limit its application in genome-wide screens. However, SB has successfully been used to screen for new cancer genes (14,15).PiggyBac was isolated from Trichoplusia ni (16) and subsequently found to transpose efficiently in many different species (5,(17)(18)(19). One important feature of the PB transposon is that it nearly always excises itself precisely and leaves no footprint behind (5,20,21). It was demonstrated that PB was very efficient for germ-line mutagenesis in the mouse (22, 23). It was subsequently confirmed that PB has significantly higher transposition activity in mammalian cell lines than SB and Tol2 (24). These studies suggest that PB has wide applications in dissecting gene functions (25).To facilitate PB's application in mammals, we have characterized further the properties of the PB transposon. We measured the transposition efficiencies of PB in mouse ES cells. We also constructed a PB/SB hybrid transposon to compare the PB and SB transposon systems in ES cells. Our data demonstrated that PB transposition was inhibited by DNA methylation. We then measured the excision and reintegration rates of a single PB transposon from two independent genomic loci and tested its ability to mutate a genomic locus with gene trap cassettes. Finally, we investigated PB's integration site distribution in the mouse genome and found that at least 9% of transposition eve...

Wnt Pathway Regulation of Embryonic Stem Cell Self-Renewal

“…Tcf3 was one of the 12 targets whose knockdown allowed the maintenance of Nanog-GFP levels after retinoic acid. In a separate phenotype-driven genetic screen, the Blm 2 mutator mESC system (Guo et al 2004) was combined with PiggyBAC mutagenesis (Ding et al 2005;Wang et al 2008) to identify recessive mutations that inhibited neural differentiation in a colony-based assay (Guo et al 2011). This pilot screen of 2000 random mutant mESC lines recovered five lines that failed to differentiate when stimulated; all five harbored a mutation in the Tcf3 gene and were likely identical by descent (Guo et al 2011).…”

Chromosomal mobilization and reintegration of Sleeping Beauty and PiggyBac transposons

“…Meiotic mobilization of PB has been reported to show no local reintegration bias, although studies in ES cells have observed a hot spot (Wang et al, 2008).…”

“…As expected our analysis of SB insertions revealed that 25% reinserted within 4 Mb of the Hprt donor site. PB was believed not to exhibit local hopping, although one recent study has reported an integration preference for PB on the donor chromosome with a striking local preference, with 9% of the insertions being clustered within 100 kb of the donor site (Wang et al, 2008). The transposition events analyzed by Wang et al, (2008) used a gene-trap selection cassette, which imposes a bias on the distribution of these events in the genome.…”

“…PB was believed not to exhibit local hopping, although one recent study has reported an integration preference for PB on the donor chromosome with a striking local preference, with 9% of the insertions being clustered within 100 kb of the donor site (Wang et al, 2008). The transposition events analyzed by Wang et al, (2008) used a gene-trap selection cassette, which imposes a bias on the distribution of these events in the genome. Our analysis of PB insertions excised from Hprt under nonselective conditions did not identify integration preferences, although reintegrations in the Hprt locus, which extends over 40 kb, would have mutated the gene and would not survive HAT selection.…”

A DNA transposon-based approach to validate oncogenic mutations in the mouse

“…The continuous jumping and random reintegration of SB transposons therefore provide the opportunity for the cDNAs to select for the optimal timing of oncogenic activation as well as for the right cell compartments. A hybrid transposon consisting of SB and PB transposons should further improve the randomness of the transposon integration sites and the transposition efficiency (31). Additionally, to reduce the time required for mutation validation, the cDNA-carrying transposons can directly be transfected into ES cells expressing the transposase.…”