Murine Retroviral Bone Marrow Transplantation Models for the Study of Human Myeloproliferative Disorders

Abstract

Human myeloproliferative diseases are common hematologic disorders characterized by clonal overproduction of maturing myeloid or erythroid cells, often caused by expression of a mutant, dysregulated tyrosine kinase (TK). These diseases can be accurately modeled in laboratory mice by the retroviral transfer of a mutant TK gene into murine hematopoietic stem and progenitor cells, followed by transplantation of these cells into irradiated recipient mice. This yields a model system for analyzing the molecular pathophysiology of these conditions and provides a platform for testing therapies, particularly molecularly targeted new chemical entities (NCEs). The Basic Protocol in this unit describes the preparation of mouse bone marrow cells to express the relevant human oncogene before transplanting them into irradiated recipient mice. An alternate protocol describes a similar technique that allows specific induction of lymphoproliferative disease by some TKs. Support protocols for generating and titering retroviral stocks are also included. Curr. Protoc.

Distinct GAB2 signaling pathways are essential for myeloid and lymphoid transformation and leukemogenesis by BCR-ABL1

“…Ecotropic retroviral stocks were generated by transient transfection of 293 cells using the kat packaging system, 10 and stocks were matched for titer by transduction of NIH3T3 cells. Transduction frequency was assessed by flow cytometric detection of GFP 34 or, for bicistronic viruses coexpressing BCR-ABL1 and GAB2, by intracellular staining for HA epitopetagged GAB2 proteins (see below), as well as by Southern blot analysis of proviral DNA content in genomic DNA. 34 Titers determined by either method were concordant.…”

“…Transduction frequency was assessed by flow cytometric detection of GFP 34 or, for bicistronic viruses coexpressing BCR-ABL1 and GAB2, by intracellular staining for HA epitopetagged GAB2 proteins (see below), as well as by Southern blot analysis of proviral DNA content in genomic DNA. 34 Titers determined by either method were concordant.…”

“…34 Transduced cells were plated in cytokine-free methylcellulose (M3234; Stem Cell Technologies) for myeloid colony assays or injected intravenously into lethally irradiated recipients for myeloid leukemogenesis, as previously described. 9 For B-lymphoid transformation/leukemogenesis, BM from Gab2 1/1 or Gab2 2/2 donors was transduced once with BCR-ABL1 retrovirus in the absence of cytokines.…”

IKK-dependent activation of NF-κB contributes to myeloid and lymphoid leukemogenesis by BCR-ABL1

“…Replication-defective ecotropic retroviral stocks were generated by transient transfection of 293 cells using the kat packaging system and titered by transduction of NIH3T3 cells, as described 20 Titers determined by both methods were concordant. Viral stocks were matched by comparing the proviral copy number induced by serial dilutions and selecting concentrations that yield equivalent transduction in primary BM cells ( Figure S1).…”

“…Induction of B-ALL and CML-like MPN in the retroviral BM transduction/transplantation model system has been described in detail elsewhere. 20 For assessment of myeloid colony formation, 1 × 10 4 transduced cells were plated in triplicate in cytokine-free methylcellulose (M3234, Stem Cell Technologies), and colonies were counted on d14. Assessment of BCR-ABL1 B-lymphoid transformation by pre-B cell colony formation in agarose 22 and stromal-dependent growth 23 was performed as described.…”