Terminal inverted repeats of the Sleeping Beauty DNA transposon possess residual promoter-like activities

Description

Sleeping Beauty (SB) DNA transposon-based vectors belong to a growing family of non-viral gene-integrating vectors that represent attractive alternatives to conventional virus-based integrating gene vehicles.

In early studies of gene transfer vectors based on the SB transposon we found indications that the terminal inverted repeat (IR)-sequences are able to drive gene expression of downstream genes. To test the hypothesis that transposon IRs can act as promoters we carried out expression studies based on reporter constructs containing the Firefly luciferase (luc) gene. In separate constructs, DNA sequences containing the left IR (LIR) and right IR (RIR) of SB were inserted in front of the reporter gene. To detect potential orientation differences LIR and RIR were tested in both ‘inwards' (pointing towards the transposon center) and ‘outwards' (pointing away from the transposon body) orientations. In initial studies carried out in HeLa cells we observed a 3-fold increase in expression compared to levels obtained with a promoter-deficient control construct. The LIR-inwards and in particular the RIR-inwards sequences were consistently found to facilitate highest levels of activity in a number of transfected cell lines.

The inverted repeats of commonly used SB-based vectors are flanked on the inner side by short sequences (about 150-bp long) derived from the transposase gene of the original Tanichthys albonubes element. To investigate a potential importance of these regions we generated deletion mutants containing only the IR sequences (defined by the terminal direct repeats in each IR). To our surprise, these shorter promoters did not support transient luc expression. To further characterize the role of this DNA segment we created a panel of 30-bp deletion mutants based on the RIR. Luc expression assays revealed the existence of both transcriptional enhancer and repressor elements in this region of the SB vector. In addition, IR-deleted variants were inactive, indicating that promoter activity relied on sequences within the IR.

The impact of IRs with residual promoter activity on expression of transgenes from integrated SB vectors is currently not known. We have demonstrated, however, that expression from SB-IRs is sufficient to drive expression of a selectable marker gene, located inside the transposon, indicating that the transposon termini of a SB vector may influence expression of its genetic cargo. 

We conclude that the SB inverted repeats possess promoter-like activities and that this activity is further enhanced by the sequences flanking the inner direct repeat of both LIR and RIR.