Figure 2.

Generation of an EVI1-GFP inv(3) AML model.

A. Schematic representation of EVI1-GFP knock-in with a T2A self-cleavage site in the MUTZ3 cells at the endogenous translocated EVI1 locus.

B. Flow cytometric analysis of intracellular EVI1 after shRNA-mediated knockdown of EVI1 using two different shRNAs. The effects on EVI1 protein were measured 48 hours after transduction. Scrambled shRNAs were used as control.

C. Flow cytometric analysis of GFP in the same experiment indicated in (B).

D. Representative flow cytometric plot showing the effect of the −110kb GATA2 enhancer deletion in MUTZ3-EVI1-GFP cells (Δ enhancer). Cas9 was induced with Dox 24h before nucleofection of two sgRNAs. The effect on EVI1 was measured by GFP levels using flow cytometric analyses. Cells were sorted 48h after nucleofection of subsequent sgRNAs into three fractions: GFP^low, GFP^mid and GFP^high.

E. Genotyping PCR showing a wild type (WT) band (1500 bp) or a band for the enhancer deleted(Δ) (900 bp), either in bulk (before sorting) or in sorted fractions. Control (Ctrl) represents PCR after nucleofection of the sgRNAs without Dox induction.

F. Bar plot showing relative GFP expression of bulk and sorted fractions analyzed by qPCR. The expression levels of PBGD, a housekeeping gene, were used as control for normalization. Relative expression is calculated as fold over Ctrl (nucleofection of the sgRNAs without Dox). Error bars represent standard deviation of two biological replicates.

G. Bar plot showing relative EVI1 expression of MUTZ3-EVI1-GFP bulk and sorted fractions analyzed by qPCR. For details see Figure 2F legend.

H. Bar plot showing the number of colonies grown in methylcellulose from each sorted fraction. Colonies were counted 1.5 weeks after plating. Error bars represent standard deviation of three plates.