Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Summary

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

Abstract

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

Introduction

Transduction-transplantation is a useful method to model hematologic malignancies in mice. This technique has been particularly valuable for studying myeloid malignancies dating back to the first demonstration that ectopic expression of BCR-ABL1 could faithfully recapitulate chronic myelogenous leukemia in mice¹. This technique has subsequently facilitated the extensive study of JAK2^V617F and MPL^W515K/L mutated myeloproliferative neoplasm (MPN).

MPN are a group of hematologic malignancies characterized by the overproduction of mature myeloid cells and bone marrow fibrosis. These diseases generally arise from....

Protocol

This study was approved and carried out in accordance with the recommendations by the Institutional Animal Care and Use Committee at University of California, Irvine. All procedures were performed under isoflurane anesthesia and all efforts were made to minimize suffering.

1. Generation of Ecotropic Retrovirus

1. Prepare high-quality plasmids with a concentration of at least 1 µg/µl using either a commercial maxi-prep kit or cesium chloride purification.
   NOTE: These include an MSCV backbone vector encoding the gene of interest (in this case CALR^7,8) and marker of choice (GFP, Neo, etc.) as well as a....

Results

The transduction-transplantation technique results in hematopoietic reconstitution of the recipient mice with cells expressing the gene of interest. Figure 1 shows an overview of the transduction-transplantation mouse model of calreticulin mutated MPN. Briefly, retrovirus expressing CALRwt or CALRdel52 is used to infect BM cells from a C57B/6 donor mouse. Transduced cells are transplanted into irradiated C57B/6 recipient mouse and donor cell engraftment occurs during the .......

Discussion

This protocol provides a detailed description of how to perform bone marrow transplants in mice to recapitulate an essential thrombocythemia-like disease with progression to myelofibrosis with CALRdel52 mutation as the driver of disease. Successful transplantation of cells expressing CALRdel52 results in increased platelets, expansion of megakaryocytes, and bone marrow fibrosis. As bone marrow transplantation is a multistep process, it is important to acknowledge steps where technical error can be avoided to prevent poor.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell Lines
DMEM	Corning	MT-10-013-CV
293T cells	ATCC	CRL-11268
3T3 cells	ATCC	CRL-1658
Plasmids
EcoPak, also known as pCL-Eco	Addgene	12371	Retroviral packaging cell lines, such as EcoPack 2-293, may be used in place of the EcoPak plasmid and standard 293T cells. Additional γ-retrovirus envelope and packaging plasmids are available from Addgene and others.
MSCV-IRES-GFP (MIG)	Addgene	20672	Additional γ-retroviral transfer plasmids are available from Addgene and others.
Consumables
27 G x 1/2" needles	BD	305620
Fetal bovine serum	Corning	MT-35-010-CV
Penicillin/streptomycin/L-glutamine	Corning	MT-30-009-CI
Trypsin-EDTA (0.05%)	Corning	MT-25-052-CI	Can be homemade
PBS	Corning	MT-21-031-CV
10 cm dishes	Fisher	172931
15 ml conical tubes	Fisher	12565268
60 mm dishes	Fisher	150288
Polybrene	Fisher	NC9840454
5-FU	Fisher	A13456-06
100 µm cell strainers	Fisher	22363549
50 ml conical tubes	Fisher	12565270
6-well plate	Fisher	130184
FACS tubes	Fisher	14-959-5
0.45 μm syringe filters	Fisher	0974061B
Opti-MEM	Gibco	31985-070
ACK buffer	Lonza	10-548E	Can be homemade
Recombinant murine IL-3	Peprotech	213-13
Recombinant murine IL-6	Peprotech	216-16
Recombinant murine SCF	Peprotech	250-03
X-tremeGENE 9	Roche	6365809001	Transfection reagent
1.5 ml centrifuge tubes	USA Scientific	1615-5500
Equipment
BD Accuri C6
X-ray irradiator