Trasduzione-trapianto modello murino di neoplasia mieloproliferativa
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
Trasduzione-trapianto è un metodo utile per modellare neoplasie ematologiche nei topi. Questa tecnica è stata particolarmente utile per lo studio di neoplasie mieloidi risalenti alla prima dimostrazione che l'espressione ectopica di BCR-ABL1 potrebbe fedelmente ricapitolare leucemia mieloide cronica nei topi 1. Questa tecnica ha successivamente facilitato l'ampio studio di JAK2 V617F e MPL W515K / L mutato neoplasia mieloproliferativa (MPN).
MPN sono un gruppo di neoplasie ematologiche caratterizzate dalla sovrapproduzione di cellule mieloidi mature e fibrosi del midollo osseo. Queste malattie in genere derivano dall'espansione clonale di una cellula staminale emopoietica che ha acquisito una mutazione somatica in entrambe le Jak2, MPL, o CALR. Trasduzione-trapianto di JAK2 V617F e MPL W515K / modelli L mostra le caratteristiche cliniche della policitemia vera e mielofibrosi 2-5 </ Sup>. Di recente, un modello murino di calreticulin-mutato MPN è stato anche generato con il metodo di trasduzione-trapianto 6. Questi topi sviluppano una malattia trombocitemia essenziale simile ad un aumento delle piastrine, aumento del numero di megacariociti, e fibrosi del midollo osseo. Insieme, questi modelli non solo hanno fornito l'occasione al fine di conoscere la patogenesi molecolare di MPN, ma anche la capacità di sviluppare e studiare terapie in un ambiente pre-clinica.
Questo manoscritto fornisce una descrizione dettagliata della metodologia di trasduzione-trapianto con una particolare attenzione per la mutazione CALRdel52. Questa tecnica prevede il trapianto di cellule del midollo osseo retrovirally trasdotte che esprimono il mutante costrutto in topi riceventi singenici irradiati.
Protocol
Representative Results
Discussion
Questo protocollo fornisce una descrizione dettagliata di come eseguire trapianti di midollo osseo in topi ricapitolare una malattia essenziale trombocitemia simile con la progressione di mielofibrosi con CALRdel52 mutazione come il conducente della malattia. il trapianto di successo di cellule che esprimono i risultati CALRdel52 un aumento delle piastrine, l'espansione dei megacariociti, e fibrosi del midollo osseo. Come trapianto di midollo osseo è un processo a più fasi, è importante riconoscere passaggi dove …
Disclosures
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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