A hyperactive piggyBac transposase for mammalian applications

Abstract

DNA transposons have been widely used for transgenesis and insertional mutagenesis in various organisms. Among the transposons active in mammalian cells, the moth-derived transposon piggyBac is most promising with its highly efficient transposition, large cargo capacity, and precise repair of the donor site. Here we report the generation of a hyperactive piggyBac transposase. The active transposition of piggyBac in multiple organisms allowed us to screen a transposase mutant library in yeast for hyperactive mutants and then to test candidates in mouse ES cells. We isolated 18 hyperactive mutants in yeast, among which five were also hyperactive in mammalian cells. By combining all mutations, a total of 7 aa substitutions, into a single reading frame, we generated a unique hyperactive piggyBac transposase with 17-fold and ninefold increases in excision and integration, respectively. We showed its applicability by demonstrating an increased efficiency of generation of transgene-free mouse induced pluripotent stem cells. We also analyzed whether this hyperactive piggyBac transposase affects the genomic integrity of the host cells. The frequency of footprints left by the hyperactive piggyBac transposase was as low as WT transposase (~1%) and we found no evidence that the expression of the transposase affects genomic integrity. This hyperactive piggyBac transposase expands the utility of the piggyBac transposon for applications in mammalian genetics and gene therapy.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Hyperactive mutant screening in yeast. (A) Schematic representation of the excision assay in yeast. The URA3 gene is separated by the actin intron containing the mini-piggyBac transposon, which completely disturbs the normal splicing. After excision, the actin intron is spliced out normally and the URA3 gene is restored. (B) Relative activities of hyPBase mutants in yeast. These values are the median of assays of 10 colonies of WT and each mutant type performed in glucose, i.e., without induction of the GALS promoter. The absolute value of transposition promoted by WT transposase was 4.7 × 10−4 ura+ cells/total cells.

Fig. 2.

Comparison of the transposase mutants and generation of hyperactive transposase in mouse ES cells. (A) Schematic representation of the excision assay in ES cells. The Hprt gene is disrupted by the piggyBac transposon carrying a gene-trap unit; thus, the ES cells are sensitive to HAT. When the transposon jumps out, the Hprt gene is restored, making cells resistant to HAT. (B) Schematic representation of the integration assay in ES cells. The piggyBac transposon vector carrying a gene-trap cassette was cotransfected into WT ES cells together with a transposase expression vector. When the transposon jumps into an active gene, the puromycin resistant gene is expressed; thus cells become resistant to puromycin. White boxes, exons; SA, splice acceptor site; IRES, internal ribosome entry site; puΔtk, the puromycin-resistant gene fused with the herpes simplex virus thymidine kinase gene; pA, poly-adenylation signal. (C) Relative excision and integration activities of the hyperactive mutant in ES cells (Upper) and protein expression of HA-tagged PBase and mutants in 293T cells (Lower). As a control, a WT transposase with the original insect-derived cording sequence was used. The mutants indicated by arrows were combined for the generation of the hyperactive transposase. Representative data are shown. Asterisk marks nonspecific band. (D and E) Comparison of hyPBase with the WT transposase (mPBase) in excision (D) and integration (E) assays. Data are shown as mean ± SD (n = 3). (F) Western blot analysis showing expression of HA-tagged mPBase and hyPBase in ES cells. Dilution factors are shown on the top of gel picture. β-Actin was used for loading controls. Asterisk marks nonspecific band.

Fig. 3.

Analysis of the piggyBac excision-induced mutations. (A) Schematic representation of isolation of cells with footprints. In HprtPB_ex3 ES cells, the Hprt gene is disrupted by targeted insertion of the piggyBac transposon carrying the puΔtk cassette into a TTAA site in exon 3. When the excision site is precisely repaired, exon 3 is reconstructed and thus the Hprt gene is restored. If a footprint is generated, then the Hprt is permanently disrupted. Cells with footprints can be selected by FIAU and 6TG double selection. (B) Frequencies of Hprt-deficient colonies after transposon excision. Data are shown as mean ± SD (n = 6 for mPBase, n = 3 for hyPBase). (C) Sequences of the footprints generated after mPBase- or hyPBase-mediated excision. The donor TTAA site is highlighted in red. (D) The piggyBac excision-induced genomic alterations. Most of these carry broken transposons (the remaining part of the transposon is shown in red). A 413-bp unrelated sequence (shown in gray) was inserted in hyPBase clone 4. Redundant clones are not shown. Exon 3 with the transposon insertion is shown as E3-1 and E3-2. Red box indicates the piggyBac transposon; black boxes are exons; thin lines are the regions deleted.

Fig. 4.

Improved generation of transgene-free iPS cells using the hyPBase. (A) A representative image of alkaline phosphatase staining of iPS cell colonies generated using WT (Left) and hyperactive (Right) PBase. (B) Numbers of iPS cell colonies obtained from transfection of MEFs with 100 ng transposon and 100 ng transposase in a 12-well plate. Data are shown as mean ± SD (n = 3) (C) Number of transgene-free iPS cell colonies generated by mPBase or hyPBase. Both primary iPS cell lines (iPS25 and iPS28) have two transposon integrations. Representative data are shown. (D) PCR analysis showing transposon removal and no evidence of random integration of plasmids. (E) Precise repair of the excised site. All clones examined possess intact genomic sequences. The transposon donor sites are highlighted in red.