Gene expression changes in NRASG12D, NUP98-NSD1 and NUP98-NSD1+NRASG12D transformed mouse bone marrow cells (in vitro).

NUP98-NSD1 positive Acute myeloid leukemia (AML) frequently occurs within the pediatric karyotypic normal(CN)-AML cohort. It is often associated with mutations in genes like FLT3, NRAS, WT1 and MYC. Here we have studied the role of NUP98-NSD1 fusion and NRASG12D in leukemia initiation and progression. Overall design: After 4 days of treatment with 150 mg 5-fluorouracil/kg, bone marrow cells were harvested from mice. Cells were stimulated for 48 hours in Dulbecco's modified Eagle medium (DMEM) (STEM cell Technologies, Cologne, Germany) supplemented with 15% fetal bovine serum (FBS) (Sigma-Aldrich, Munich, Germany), 10 ng/mL human interleukin-6 (hIL-6), 6 ng/mL murine interleukin-3 (mIL-3), and 20 ng/mL murine stem cell factor (mSCF; all from PeproTech, Hamburg, Germany). Retrovirus producing Phoenix packaging cells were transfected with the required vectors and viral supernatant was used to transduce mouse bone marrow cells using 5 µg/mL protamine sulfate (Sigma-Aldrich, Seelze, Germany). After cells were expanded, they were sorted for the respective fluorochrome expression.Total RNA was isolated and sent for sequencing.