Fig. 1
From: Active recombinant Tol2 transposase for gene transfer and gene discovery applications
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