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.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

PB transposition in the mouse ES cells. (A) Schematic of the PB transposase (helper) and transposon (donor) constructs. (B) PB transposition with increasing amounts of PB transposon. A fixed amount of CAGG-PBase was coelectroporated with increasing amounts of PB-PGK-Neo-bpA donor plasmid. Each number is the average obtained from three independent experiments. Error bars indicate the standard deviation from the mean. (C) PB transposition with increasing amounts of PB transposase. A fixed amount of PB-PGK-Neo-bpA transposon was coelectroporated with increasing amounts of CAGG-PBase helper plasmid. (D) Southern analysis of PB transposition events. Twelve G418R colonies were picked from each experiment. There is a unique EcoRV site in the PB-PGK-Neo-bpA transposon. Genomic DNA was digested with EcoRV and hybridized with a Neo probe. Variation of the EcoRV fragment size illustrates independent PB transposition events.

Fig. 2.

Direct comparison of PB and SB transposases, using the PB/SB hybrid transposons. (A) Schematic of PB/SB hybrid transposons, PBase, and SB11 transposase knockin cells lines. (Upper) PB-SB-PGK-Neo-bpA and PB-SB-SA-βgeo hybrid transposons. (Lower) Schematic diagram of the Rosa-PBase and Rosa-SB11 alleles. (B) Transposition efficiencies of the PB-SB-PGK-Neo-bpA hybrid transposon in Rosa-PBase and ROSA-SB11 knockin cell lines. SB transposition efficiency (1 μg of PB-SB-PGK-Neo-bpA) was arbitrarily used as the standard. There was a 1,310-fold difference between these two lines (P < 0.002) when 1 μg of the transposon DNA was used. The fold change dropped to 675-fold (P < 0.001) when 10 μg of the PB-SB-PGK-Neo-bpA transposon plasmid was used. (C) Southern blot analysis of transposition events in Rosa-PBase and Rosa-SB11 knockin lines. Eight G418 resistant colonies were picked from the PB or SB transposition experiments. Genomic DNA was digested with EcoRV and hybridized against a Neo probe. The 5-kb EcoRV fragment present in each clone comes from the insertion of a nonfunctional Neo cassette at the Hprt locus in AB2.2 cells. Other fragments represented independent transposition events. (D) Methylation of the transposon enhanced SB-mediated transposition. Methylated and unmethylated PB-SB-SA-βgeo DNA was electroporated into Rosa-SB11 cells. The trapping efficiency was calculated as the percentage of G418R cells in all of the cells surviving the electroporation. Relative trapping efficiency was normalized to the trapping efficiency of the unmethylated transposon; P < 0.02. (E) Methylation of the transposon inhibited PB-mediated transposition. The same experiment was performed in Rosa-PBase cells as in D; P < 0.002.

Fig. 3.

Re-mobilization of PB/SB hybrid transposon. (A) Illustration of the PGK-[PB-SB-SA-βgeo-PGK-Bsd]-Puro knockin allele at the Rosa26 locus. A SA-βgeo trapping cassette and a PGK-Bsd selection cassette were cloned into the PB/SB hybrid transposon to form the PB-SB-SA-βgeo-PGK-Bsd transposon. This complex transposon was subsequently inserted in between the PGK promoter and the Puro coding sequence in a PGK-Puro cassette and targeted to the Rosa26 locus. Transfection of the targeted ES cells with CAGG-PBase plasmid mobilized the targeted transposon. Excision reunited the PGK promoter and Puro coding sequence so that the excision events can be scored by puromycin resistance. (B) Transposon excision rates from the Rosa26 and Hprt loci. CAGG-PBase DNA were electroporated into Rosa-PGK-[PB-SB-SA-βgeo-PGK-Bsd]-Puro targeted cells to mobilize the transposon. The excision rate was adjusted to account for all of the cells surviving the electroporation. Similar experiments were carried out in the cells that the PB-SB-SA-βgeo transposon inserted into the Hprt locus (see Fig. 4). (C) Southern blot analysis of the excision events from the PGK-[PB-SB-SAβgeo-PGK-Bsd]-Puro knockin cells. The original targeted clone had both the 3.1-kb targeted EcoRV band and the 5.3-kb transposon fragment. Clones that lost PB after excision only have the 3.8-kb excision fragment. Clones in which transposon excision was followed by reintegration had both the 3.8-kb excision and 5.3-kb transposon fragments.

Fig. 4.

PB-mediated mutagenesis. (A) Schematic illustration of a PB-SB-SA-βgeo transposon integrated into the Hprt locus. AB1 ES cells have one copy of Hprt gene. If a PB-SB-SA-βgeo transposon integrates into Hprt locus and disrupts its function, AB1 ES cells will become HAT sensitive and 6-TG resistant. If this transposon is remobilized, the cells will become HAT resistant and 6-TG sensitive. (B) Transposon insertions in the Hprt locus. Transposon-genomic junction fragments from five 6-TG clones were mapped to the Hprt locus. Filled boxes, Hprt exons; white arrowhead, transposon integration sites of clones in which the Hprt exons were directly spliced the SA-βgeo cassette; black arrowhead, the transposon integration site of a clone in which the Hprt gene was disrupted by PB 5′TR. (C) Nearly precise excision of the PB transposon from the Hprt and Rosa26 loci. PCR products that amplify the excision sites in 30 independent excision events from the Hprt locus and 32 from the Rosa26 locus were sequenced. All but three excision events were precise. One clone had a 4-bp microdeletion, another had a 1-bp deletion, and the third had a 6-bp microdeletion.

Fig. 5.

Distribution of PB integration sites in the genome. (A) PB integration sites in the mouse genome from random transposition events. The sites were identified from transposition experiments, using CAGG-PBase and PB-SB-PGK-Neo-bpA. The transposon-genomic junction fragments were mapped to the mouse genome and displayed in Ensembl (version 48.37a). (B) PB integration sites of gene trapping clones. Splinkerette PCR was performed on G418R clones from coelectroporation of CAGG-PBase and PB-SB-SA-βgeo. Transposon-genomic junction fragments were then mapped to the mouse genome. (C) Local hopping of PB transposition from the Rosa26 locus. Nine percent of PB reintegration sites (21/264) were clustered within a 100-kb region flanking the Rosa26 locus on the donor chromosome (chr. 6). The rest of the integration sites appeared to be randomly distributed in the genome. The height of the peaks on the histograph represent the number of integration sites in a genomic region. (D) No apparent local hopping of the PB transposition from the Hprt locus. A total of 93 integration sites were cloned from PB transposition from the Hprt locus. Analysis of these sites did not show any obvious bias to either the Hprt locus or the X chromosome.