LOF-CD44 and GOF-CD44 BMC are generated using retroviral transduction.

<p>CD44^-/- BMC were isolated from the femurs and tibia of mice that had received 5-FU 4 days prior, and were then cultured with 3/6/SCF medium. CD44^-/- BMC were transduced with GOF-CD44 or LOF-CD44 constructs, and CD44^+/+ BMC were transduced with an empty vector (WT). (A) FC plots of GFP or RFP expression versus CD44 expression. (B) FC plots of FL-HA binding versus CD44 expression of WT, CD44^-/-, LOF-CD44 or GOF-CD44 transfected BMC. (C) Histograms and mean fluorescence intensity (MFI) of CD44 expression of WT, LOF-CD44 or GOF-CD44 expressing BMC, after gating on reporter-positive BMC. Representative of two experiments. (D) Percentage of lineage^- and LSK cells within total, untransduced and transduced BMC. Mean +/- SD from eight biological replicates of three independent experiments. **p<0.01 calculated by Student’s t-test.