Transduction-Transplantation Modèle de souris de syndrome myéloprolifératif
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 est une méthode utile pour modéliser les hémopathies malignes chez les souris. Cette technique a été particulièrement précieux pour l' étude des tumeurs malignes myéloïdes remontant à la première démonstration que l' expression ectopique de BCR-ABL1 pourrait fidèlement récapituler la leucémie myéloïde chronique chez les souris 1. Cette technique a ensuite facilité l'étude approfondie de JAK2 V617F et MPL W515K / L muté syndrome myéloprolifératif (MPN).
NPP sont un groupe de tumeurs malignes caractérisées par une surproduction de cellules myéloïdes matures et une fibrose de la moelle osseuse. Ces maladies proviennent généralement de l'expansion clonale d'une cellule souche hématopoïétique qui a acquis une mutation somatique soit dans Jak2, MPL ou CALR. Transduction-transplantation JAK2 V617F et MPL W515K / L modèles présentent les caractéristiques cliniques de la maladie de Vaquez et myélofibrose 2-5 </ Sup>. Récemment, un modèle de souris de la calréticuline mutée MPN a été généré par la méthode de transduction transplantation 6. Ces souris développent une maladie thrombocytémie comme essentielle à l'augmentation des plaquettes, l'augmentation du nombre de mégacaryocytes et la fibrose de la moelle osseuse. Ensemble, ces modèles ont non seulement donné l'occasion de mieux comprendre la pathogenèse moléculaire de MPN, mais aussi la capacité à développer et à étudier la thérapeutique dans un cadre pré-clinique.
Ce manuscrit fournit une description détaillée de la méthodologie transduction-transplantation avec un accent sur la mutation CALRdel52. Cette technique implique la transplantation de cellules de moelle osseuse transduites par un retrovirus exprimant le mutant de construction dans des souris receveuses syngéniques irradiés.
Protocol
Representative Results
Discussion
Ce protocole fournit une description détaillée de la façon d'effectuer des greffes de moelle osseuse chez des souris de récapituler une maladie thrombocytémie comme essentielle à la progression de la myélofibrose avec mutation CALRdel52 comme le conducteur de la maladie. transplanter avec succès des cellules exprimant les résultats CALRdel52 une augmentation des plaquettes, l'expansion des mégacaryocytes et la fibrose de la moelle osseuse. Comme greffe de moelle osseuse est un processus en plusieurs é…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work is supported by the V Foundation Scholar (AGF) and the MPN Research Foundation (AGF).
Materials
| 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 | |||
| 27G 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 | |
| 10cm dishes | Fisher | 172931 | |
| 15 ml conical tubes | Fisher | 12565268 | |
| 60mm dishes | Fisher | 150288 | |
| Polybrene | Fisher | NC9840454 | |
| 5-FU | Fisher | A13456-06 | |
| 100um cell strainers | Fisher | 22363549 | |
| 50 ml conical tubes | Fisher | 12565270 | |
| 6-well plate | Fisher | 130184 | |
| FACS tubes | Fisher | 14-959-5 | |
| 0.45um 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 |
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