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Scheme used to identify FLT3/ITD resistance mutations. XL1-Red cells were transformed with the cFUGW plasmid carrying a human FLT3/ITD and grown overnight in agarose. The harvested DNA was used to transduce hematopoietic cells, which were then selected for resistance based on their ability to grow colonies in methylcellulose in the presence of a FLT3 TKI for 2 weeks. The resulting colonies were expanded in liquid culture growth medium, analyzed and sequenced for FLT3 mutations.
