Active recombinant Tol2 transposase for gene transfer and gene discovery applications

Abstract

Background: The revolutionary concept of "jumping genes" was conceived by McClintock in the late 1940s while studying the Activator/Dissociation (Ac/Ds) system in maize. Transposable elements (TEs) represent the most abundant component of many eukaryotic genomes. Mobile elements are a driving force of eukaryotic genome evolution. McClintock's Ac, the autonomous element of the Ac/Ds system, together with hobo from Drosophila and Tam3 from snapdragon define an ancient and diverse DNA transposon superfamily named hAT. Other members of the hAT superfamily include the insect element Hermes and Tol2 from medaka. In recent years, genetic tools derived from the 'cut' and 'paste' Tol2 DNA transposon have been widely used for genomic manipulation in zebrafish, mammals and in cells in vitro.

Results: We report the purification of a functional recombinant Tol2 protein from E.coli. We demonstrate here that following microinjection using a zebrafish embryo test system, purified Tol2 transposase protein readily catalyzes gene transfer in both somatic and germline tissues in vivo. We show that purified Tol2 transposase can promote both in vitro cutting and pasting in a defined system lacking other cellular factors. Notably, our analysis of Tol2 transposition in vitro reveals that the target site preference observed for Tol2 in complex host genomes is maintained using a simpler target plasmid test system, indicating that the primary sequence might encode intrinsic cues for transposon integration.

Conclusions: This active Tol2 protein is an important new tool for diverse applications including gene discovery and molecular medicine, as well as for the biochemical analysis of transposition and regulation of hAT transposon/genome interactions. The measurable but comparatively modest insertion site selection bias noted for Tol2 is largely determined by the primary sequence encoded in the target sequence as assessed through studying Tol2 protein-mediated transposition in a cell-free system.

Keywords: Recombinant transposase protein; Tol2 transposase; Transposition site preference; Zebrafish; hAT superfamily.

Fig. 1

The highest activity Tol2 isoform and its modification by 6X His tag. a Tol2 genomic DNA (GenBank: D84375.2) structure and expressed mRNA from various sources (Tol2-S GenBank: AB031080; Tol2-L GenBank: AB031079; Tol2-M GenBank: AB032244). The first nucleotide location is numbered in relation to genomic DNA sequence in each mRNA and indicated by an open-ended arrow. The start and stop codons of the longest open reading frame in each mRNA are also indicated. Tol2-L spans all 4 exons (black bar, E1-4) while Tol2-S covers only the last three (E2-4). Tol2-M starts further into exon 1 than L. The predicted Tol2-M protein is slightly shorter at the amino-terminus than Tol2-L. Corresponding sequences encoding the BED zinc finger DNA binding motif are indicated on genomic DNA and mRNA. b In this paper we used a modified Tol2-M mRNA [13] encoding the same protein as Tol2-M, but all the nucleotides 5′ to the first start codon in Tol2-M (Fig. 1a, Tol2-M gray box) were deliberately removed to eliminate any possible effects from an out-of-frame ATG within those sequences. 6X His tags were added to either 5′- or 3′ of the modified mRNA, and the deduced N-terminus or C-terminus modified Tol2 protein sequences were outlined. c Human codon-optimized Tol2-M was most effective for gene transfer in HeLa cells. Tol2-M was tested for activity in human cell culture by colony forming assay. A Tol2 transposon vector carrying a Zeocin resistance gene cassette was co-transfected with different version of Tol2 or GFP control driven by CMV promoter at 50 ng or 500 ng. The numbers of Zeocin-resistant colonies formed in each treatment were compared to the treatment with the highest colony numbers (500 ng CMV-hTo2-M) as percentages. Bars represent mean values ± SEM of three independent experiments. * or ** indicate data is significantly different from respective CMV-GFP controls (* t-test P < 0.05; ** t-test P < 0.01). d Tol2 transposase could be modified by 6XHis epitope without loss of activity. A GFP-reporter [13] was co-injected with synthesized mRNA corresponding to untagged Tol2 or Tol2 tagged with 6X-His at either N-terminus (His-Tol2) or C-terminus (Tol2-His). Somatic GFP expression was scored at 3 dpf and the percentages of injected fish demonstrating GFP only in the body (body only) or demonstrating strong GFP in eyes and anywhere in the body (body + eyes) were recorded

Fig. 2

Expression and detection of recombinant Tol2 transposase protein. a Tol2 transpoase sequence was cloned into an N-terminal 6XHis expression vector pET-21a (Novagen). The expression cassette driven by T7 promoter is shown, and a fusion protein of ~74 kD was expected. rbs: ribosome binding site. b Expression of His-Tol2 in E.coli BL21-AI strain. unind: cell lysate from uninduced cells harboring Tol2-expression vector; t: total induced crude cell lysate; i: insoluble protein fraction from induced cell lysate; s: soluble protein fraction from induced cell lysate. Equal volume of cell culture was loaded in each lane. p: ~ 350 ng purified His-Tol2 protein (arrow head). c Immunoblot analysis of His-Tol2 expression with anti-His antibody. The same protein samples as in (b) were loaded except that only ~ 6 ng purified His-Tol2 was used for detection by western blot

Fig. 3

His-Tol2 is a fully functional transposase in vivo. a Diagram showing microinjection of zebrafish embryo at one-cell stage to generate transgenic animals. GBT-RP2 plasmid containing GFP-reporter gene trap was co-injected with either Tol2 mRNA or His-Tol2 protein. Mosaic somatic GFP signals were seen in F0 injected fish. F0 embryos were raised to adult and outcrossed to obtain F1 generation. Ubiquitous GFP would be detected in F1 fish if the integration was due to a gene trap event and was passed through the germline. Molecular analysis of transposon insertion numbers and genomic locations was carried out on tissue from F1 generation animals. P_β-actin: β-actin promoter; SD: splice donor. b Representative images of F0 embryos demonstrating a catalog of GFP-positive somatic patterns that could be observed at 3 dpf from microinjections of different methods. Notice the signal difference in eyes (double arrow) and brain regions (open end arrow) for each category. With vector-only injections, notice only Cat. I and II patterns were observed. c A small number of His-Tol2 protein injected embryos showing ubiquitous whole body green fluorescence as early as 24 hpf. Those fish generally displayed uniform GFP expression throughout the body, with few uneven myotome GFP stripes at 3 dpf (Cat. IV). Also shown are 3 dpf injected fish somites at higher magnification, demonstrating the difference in uniformity of GFP signals in tissues from various injection categories. Injection GFP pattern distribution from typical RNA or protein mediated injection was shown. d GFP-positive F1 fish from either founder family generated by mRNA injection or His-Tol2 injection were subjected to fin-clip and Southern blot analysis with a GFP probe. Each lane represents one individual F1 adult fish. Southern blots from 5 adult fish selected from random founder family are shown for each method. See also Additional file 1

Fig. 4

Tol2 in vitro integration assay. a Scheme of in vitro integration assay. b One example miniTol2-Kan ^R integration, revealing different kanamycin-resistant colony recovery from assays with versus without His-Tol2 protein. The amount of transformed bacteria spread on each plate is indicated. c Tol2-mediated miniTol2-Kan ^R integration. The amount of target plasmid (pGL) was kept at 0.5 pMol, while PCR fragments were mixed at three different ratios. The ratio of colony numbers (Additional file 3) recovered from LB-Kan⁺ to LB-Amp⁺ was used as an indicator of integration activity. Bars represent mean values ± SEM from three independent experiments. d Plasmids isolated from colonies on LB-Kan⁺ plate were sequenced at both junctions of miniTol2-Kan ^R insertion. 8-bp target plasmid duplications were indicated. Six examples of the integration junction are shown here

Fig. 5

miniTol2 insertion sites and consensus sequence. a Tol2-mediated insertions from four independent experiments were mapped to target plasmid (pGL) for miniTol2-Kan ^R (n = 266). Y-axis indicates the number of insertion events and orientation (positive: sense orientation; negative: anti-sense orientation) Major features of the pGL vector were also indicated. Red lines below the sequences indicate insertion “hot spots”, defined as the same locations that are discovered in more than one experiment, regardless of the insertion orientation or locations in one experiment that have transposons inserted in both directions (also see Additional file 5). b Tol2 integration site motif analyzed by WebLog (version 3.0) (n = 266) and aligned to a previous indentified weak AT-rich consensus in vivo [12]