Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Hyperactive sleeping beauty transposases for enhanced utility in gene transfer in vertebrates

By using a limited site-directed mutagenesis screen, we identified hyperactive versions of the SB transposase. Three different approaches were undertaken for the choice of induced mutations:
- Modification of a linker region that separates the DNA-binding and catalytic domains;
- Systematic replacement of acidic residues with basic amino acids; and
- Substitution for amino acids that had been selected in nature.

Most of the mutations that we introduced into the SB transposase resulted in a decrease in the efficiency of transposition, suggesting very little functional redundancy in the transposase sequence. Nevertheless, one of the substitutions, the D260K mutation, produced a hyperactive phenotype. The aspartic acid in position 260 is either lysine or arginine in other Tc1-like transposases, suggesting that lysine and arginine can better function in that sequence context. It is possible, that a particular version of fish Tc1-like transposases did contain K or R at position 260, but this amino acid got replaced at some point during transposase evolution, because it is functionally non-essential for the transposase.

The D260K mutation acts synergistically with two other, naturally occurring mutations, R115H and R143C. The R115H/D260K and R115H/R143C double mutants exhibited about 3.7- and 3.2-fold increase in transposition activity over wild-type transposase, respectively. Importantly, hyperactivity of the R115H/D260K mutant (referred to as SB12) displayed additive effects with a hyperactive transposon vector. The collective effect of these components is an approximately 8-fold increase in transposition, compared to the first-generation SB system. We find that about 2% of cells that had taken up transposon DNA will undergo a transposition event using the first-generation SB system, whereas stable transgenesis rates are about 10-15% by using combinations of the hyperactive transposon/transposase components.

Transposition can be further optimized by systematic adjustment of transposase and transposon concentrations in transfected cells, because different ratios of the transposase expression- and transposon donor plasmids can greatly influence transposition efficiencies.


Zoltan IVICS, (Group leader)
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Fax: +49-309-4062547
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