Generation of an inducible and optimized piggyBac transposon system

Abstract

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.

Figure 1.

Comparison of transposase activity of the native and codon optimized piggyBac. (A) Schematic representation of the plasmids used in this experiment. The donor plasmid, 5′-PTK-3′, contains the minimal PB terminal repeats (5′-TR and 3′-TR) flanking a promoter-less puromycin resistance cassette. CMV, cytomegalovirus promoter; ATG, start codon; pA, bovine growth hormone polyA signal; En2SA, mouse engrailed-2 gene splice acceptor; IRES, internal ribosomal entry site from encephalomyocarditis virus; puΔTK, synthetic fusion CDS between the puromycin-resistance gene and a truncated thymidine kinase gene (21). (B) 2 μg of 5′-PTK-3′ and 2 μg of the transposase plasmid were co-electroporated into 10⁷AB2.2 mouse ES cells and, after puromycin selection, the numbers or resistant colonies were counted. (C) A fixed amount (1 μg) of transposase plasmid was co-electroporated with increasing amounts (1–40 μg) of 5′-PTK-3′ into 5 × 10⁶ AB2.2 ES cells and puromycin-resistant colonies were counted. (D) A fixed amount (1 μg) of 5′-PTK-3′ transposon plasmid was co-electroporated with increasing amounts (1–40 μg) of transposase plasmid into 5 × 10⁶ AB2.2 ES cells. Numbers of puromycin-resistant colonies are indicated. n = 3, error bars: SEM.

Figure 2.

Levels of protein production provided by the native and codon optimized piggyBac. (A) Schematic representation of the plasmids used in this experiment. HA, sequence encoding the hemagglutinin epitope. (B) pcDNA3-KzHA, HA-iPB or HA-mPB were transfected into COS-7 cells and the levels of HA-tagged PB transposase were analysed by Western blot using a specific monoclonal antibody raised against the HA epitope. The same membrane was subsequently incubated with a monoclonal anti-actin antibody, as loading control. Three independent transfections were performed and analysed per plasmid.

Figure 3.

Analysis of the properties of the piggyBac terminal repeats. (A) Left, schematic representation of transposon plasmids used in this experiment. Each plasmid contains the same gene trap puromycin resistance cassette flanked by different combinations of 5′-TR (blue arrows) and 3′-TR (red arrows). Right, 2 μg of transposon plasmids were co-electroporated into 10⁷ AB2.2 mouse ES cells with 2 μg of mPB helper plasmid. The numbers of puromycin-resistant colonies were counted. (B) Left, schematic representation of the firefly luciferase expression plasmids used in this experiment. 5′-TR-Luc2 contains the minimal left terminal PB repeat followed by the firefly luciferase CDS (Luc2). 3′-TR-Luc2 contains the minimal right terminal PB repeat followed by Luc2. pA, SV40 late polyA region. Right, 5′-TR-Luc2 or 3′-TR-Luc2 were transfected in HeLaS3 cells together with pGL4.74, a transfection control plasmid containing the renilla luciferase gene under the thymidine kinase promoter. Twenty-four hours after transfection firefly and renilla luciferase activities were measured. n = 3, error bars: SEM.

Figure 4.

Activity and inducibility of piggyBac transposases regulatable by 4-hydroxytamoxifen. (A) Schematic representation of the plasmids used. A modified version of the oestrogen receptor ligand-binding domain, ERT2 (orange oval), was cloned at the 5′ or 3′ of the codon optimized PB transposase CDS, to create ERT2-mPB and mPB-ERT2 plasmids. To facilitate domain folding and function, three different types of sequences encoding linker peptides were alternatively used (L1, green box; L2, blue box and L3, red box), creating six different fusion proteins. (B) 5 μg of 5′-PTK-3′ and 5 μg of the indicated transposase expressing plasmid were co-electroporated into 10⁷ AB2.2 mouse ES cells. Puromycin-resistant clones were counted following selection in the presence (+) or absence (−) of 1 μM 4-OHT. iPB and mPB were used as positive controls and pcDNA3 was used as negative control. A representative picture of plates with puromycin-resistant colonies obtained using mPB-L3-ERT2 as helper plasmid in the absence (left plate) or presence (right plate) of 4-OHT is shown. n = 3, error bars: SEM.