Introduction of Trp53 mutations into bone marrow progenitors of del(5q) MDS mouse models leads to leukemic transformation in Csnk1a1–/+ p53mut mouse. (A) Schematic experimental design: WT recipients received transplantation with Mx1-Cre+, Csnk1a1–/+, Egr1–/+, and Csnk1a1–/+Egr1–/+ bone marrow progenitor cells (ckit+) transduced ex vivo with p53-sgRNA+Cas9 or ntg-sgRNA+Cas9. (B) Frequency of mice alive after 156 days. (C) Differential blood counts in MGG stained blood smears of recipient mice at the time of euthanization (156 days after transplant). (D) MGG staining of peripheral blood smear and bone marrow cytospins of leukemic mouse (00-1). Scale bars, 10 μm in blood smear and 50 μm in bone marrow cytospin. (E) Leukemic mouse 00-1 with peripheral blood blasts exhibited splenomegaly but no thymus tumor. Scale bar denotes 10 mm. (F) Pie charts showing indel distribution among mice with matched samples from peripheral blood, sampled 4 weeks after transplantation (left pie chart), and bone marrow was sampled at harvest (right pie chart). Genomic DNA was extracted, Trp53 amplified, and Sanger sequencing performed. Indel distribution was inferred using Tracking of Indels by Decomposition decomposition of sequence traces. Wild-type Trp53 sequence are in purple, and indels of different bp length are in other colors. (G) Flow cytometry of bone marrow of leukemic mouse (00-1) and nonleukemic control mouse (99-1) shows accumulation of large lineage negative CD48+ blasts and depletion of lineage markers expressing differentiated cells. (H) Sanger sequencing of Trp53 amplicon in leukemic mouse (00-1) bone marrow cells reveals 13bp deletion within DNA-binding domain of Trp53 compared with unedited donor bone marrow sequence (BM before transduction with Trp53 sgRNA-Cas9 construct) and Trp53 amplicon of nonleukemic control mouse 99-1. BM, bone marrow.
