piggyBac is a flexible and highly active transposon as compared to sleeping beauty, Tol2, and Mos1 in mammalian cells

Abstract

A nonviral vector for highly efficient site-specific integration would be desirable for many applications in transgenesis, including gene therapy. In this study we directly compared the genomic integration efficiencies of piggyBac, hyperactive Sleeping Beauty (SB11), Tol2, and Mos1 in four mammalian cell lines. piggyBac demonstrated significantly higher transposition activity in all cell lines whereas Mos1 had no activity. Furthermore, piggyBac transposase coupled to the GAL4 DNA-binding domain retains transposition activity whereas similarly manipulated gene products of Tol2 and SB11 were inactive. The high transposition activity of piggyBac and the flexibility for molecular modification of its transposase suggest the possibility of using it routinely for mammalian transgenesis.

Fig. 1.

Schematic representations of the two-component transposon systems. (A) Helper plasmids. The expression of four transposases was driven by the CMV promoter. The plasmid backbone for all transposases is pcDNAΔneo except SB11, which was cloned in pCMV-SB11. All transposases are wild type except SB11, which is a hyperactive version. (B) Donor plasmids. A cassette with hygromycin and kanamycin resistance genes and a bacterial ColE1 replication origin was subcloned into the donor plasmid of the four transposons.

Fig. 2.

Transposition activity of SB11, Mos1, piggyBac, and Tol2 transposons in different mammalian cells. A total of 1 × 10⁵ cells per individual well in 24-well plates were transfected with 200 ng of donor plus 200 ng of helper plasmid. The pcDNA3.1Δneo vector served as a control for the absence of transposase. Transposition activity was measured by counting hygromycin-resistant colonies after a 2-week selection period. Data are shown as mean values with SD (n = 3). (A) HeLa cells. (B) H1299 cells. (C) HEK293 cells. (D) CHO cells. (E) An example of HEK293 cells transfected with piggyBac, Tol2, and SB11 transposon systems and their controls. Colonies were stained with methylene blue after 2 weeks of hygromycin selection. (F) Excision assays in HEK293. Shown is PCR analysis of excision assays performed in HEK293 transfected with donor plus helper or the control (pcDNA3.1Δneo).

Fig. 3.

The transposition activity of SB11 (A), Tol2 (B), and piggyBac (C and D) at various ratios of donor and helper. Transposition activity was measured under a fixed amount of donor plasmid (200 ng in A–C and 50 ng in D) with increasing amounts of helper plasmid cotransfected into HEK293 cells. pcDNA3.1Δneo was used to adjust the total amount of DNA transfected in each sample. Data are shown as mean values with SD (n = 3).

Fig. 4.

Transposition activity of GAL4 transposases. (A) A schematic representation of engineered chimeric transposases. A GAL4 DBD was fused in-frame to the N terminus of transposases, with a linker of 18–21 aa placed between GAL4 DBD and the transposases to facilitate proper domain folding. (B) The percentage of transposition activity relative to wild-type transposase in HEK293 cells. To compare the activity of each chimeric transposase to its wild-type counterpart, the wild-type transposition efficiency was normalized to 100%. Data are shown as mean values with SD (n = 6).