Open Access

Katharina Wächter, Eric Kowarz, Rolf Marschalek

• Our focus is the identification, characterisation and functional analysis of different MLL fusions. In general, MLL fusion proteins are encoded by large cDNA cassettes that are difficult to transduce into haematopoietic stem cells. This is due to the size limitations of the packaging process of those vector-encoded RNAs into retro- or lentiviral particles. Here, we present our efforts in establishing a universal vector system to analyse different MLL fusions. The universal cloning system was embedded into the backbone of the Sleeping Beauty transposable element. This transposon has no size limitation and displays no integration preference, thereby avoiding the integration into active genes or their promoter regions. We utilised this novel system to test different MLL fusion alleles (MLL-NEBL, NEBL-MLL, MLL-LASP1, LASP1-MLL, MLL-MAML2, MAML2-MLL, MLL-SMAP1 and SMAP1-MLL) in appropriate cell lines. Stable cell lines were analysed for their growth behaviour, focus formation and colony formation capacity and ectopic Hoxa gene transcription. Our results show that only 1/4 tested direct MLL fusions, but 3/4 tested reciprocal MLL fusions exhibit oncogenic functions. From these pilot experiments, we conclude that a systematic analysis of more MLL fusions will result in a more differentiated picture about the oncogenic capacity of distinct MLL fusions.