DNA-based transposons are natural gene delivery vehicles. Similarly to retroviruses, these elements -integrate into the chromosomes of host cells, but their life-cycle does not involve reverse transcription and they are not infectious. Transposon-based gene delivery has several advantageous features compared to viral methods; however, its efficacy has been the bottleneck of transposon utilization. Recently, using a novel strategy for in vitro evolution, we created a new hyperactive version (SB100X) of the vertebrate-specific Sleeping Beauty (SB) transposase. SB100X, when coupled with enhanced inverted terminal repeat structure T2 type SB transposons, is over 100-fold more active in mammalian cells than the prototype. We established protocol for SB100X mediated rodent transgenesis resulting on the average 35% transgenic founders with a low average number (1-2) of transgene insertions per founder. Due to these characteristics the SB100X based protocol opens the possibility of designing SB based transgenes also for in vivo knockdown experiments. By the same token, single copy transgene units introduced by the SB transposon system, more than being less prone to transgene silencing, also allow better control of transgene expression levels and patterns.