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. 2018 Jun 1;32(11-12):849–864. doi: 10.1101/gad.307504.117

Figure 2.

Figure 2.

Crlf2 and mutant Jak2 cooperate to induce murine B-ALL development in vivo. (A) Schematic of the Eµ-Crlf2 transgene. (B) Schematic overview of a fetal liver transplantation mouse model of leukemia. Fetal liver cells isolated at embryonic day 13.5 (E13.5) from Eµ-Crlf2 and wild-type C57BL/6 CD45.2+ mice were transduced with pMSCV-IRES-GFP (MIG), MIG-Jak2WT, MIG-Jak2R683G, or MIG-Jak2P933R and transplanted into sublethally irradiated (9.0 Gy) wild-type C57BL/6 CD45.1+ recipients. The resulting primary leukemias were collected and retransplanted into secondary wild-type C57BL/6 CD45.1+ recipients to derive a transplantation model for subsequent functional in vivo experiments. (C) Kapler-Meier survival curves of cohorts of wild-type C57BL/6 CD45.1+ mice transplanted with primary Eμ-Crlf2/Jak2R683G (M#9; n = 8) and Eμ-Crlf2/Jak2P933R (M#8; n = 6) cells. (D) At terminal disease or the conclusion of the experiment, mice in C were autopsied, and splenic tumor burden was assessed by weight. (E) Hematoxylin–eosin (H&E)-stained spleen and liver sections from moribund wild-type recipient mice transplanted with primary Eμ-Crlf2/Jak2R683G and Eµ-Crlf2/Jak2P933R cells. Bars, 100 µm. (F) Light microscopy of May-Grunwald-Giemsa-stained peripheral blood smears from moribund secondary recipient mice of Eµ-Crlf2/Jak2P933R cells show marked numbers of medium to large blast cells with high nuclear to cytoplasmic ratio. (G) Flow cytometry analysis of Crlf2 and GFP expression on splenocytes isolated from C57BL/6 wild-type mice and moribund secondary recipient mice transplanted with Eμ-Crlf2/Jak2R683G cells. A representative flow cytometry plot is shown. (H) Immunoblotting was performed against the indicated targets using lysates isolated from nontransformed C57BL/6 wild-type splenocytes, GFP+ Eμ-Crlf2/Jak2R683G, and Eμ-Crlf2/Jak2P933R cells. Hsp90 served as loading control. (I) Nontransformed C57BL/6 wild-type splenic B-cells, GFP+ Eμ-Crlf2/Jak2R683G, and Eμ-Crlf2/Jak2P933R cells were assayed by PCR of genomic DNA (gDNA) for VDJ rearrangements using the indicated V gene family-specific VH primers. (J) Flow cytometric analysis of the GFP+ Eμ-Crlf2/Jak2R683G cell population reveals a pre-B-cell immunophenotype (B220lowIgM−IgD−CD43−CD24+ckit+CD11b−CD3−).