Transduktion-Transplantation Mouse Model af myeloproliferative Neoplasma
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
Transduktion-transplantation er en nyttig metode til at modellere hæmatologiske maligniteter hos mus. Denne teknik har været særdeles værdifuldt for at studere myeloide maligniteter går tilbage til den første demonstration af, at ektopisk ekspression af BCR-ABL1 trofast kunne rekapitulere kronisk myeloid leukæmi i mus 1. Denne teknik har efterfølgende lettet omfattende undersøgelse af JAK2 V617F og MPL W515K / L muteret myeloproliferativ neoplasme (MPN).
MPN er en gruppe af hæmatologiske maligniteter er karakteriseret ved overproduktion af modne myeloide celler og knoglemarv fibrose. Disse sygdomme generelt skyldes den klonale udvidelse af en hæmatopoietisk stamcelle, der har erhvervet en somatisk mutation i enten Jak2, MPL eller CALR. Transduktion-transplantation JAK2 V617F og MPL W515K / L-modeller udviser de kliniske funktioner i Polycytæmia Vera og myelofibrose 2 – 5, </ Sup>. For nylig er en musemodel for calreticulin-muterede MPN også blevet genereret med transduktionen-transplantation metode 6. Disse mus udvikler en væsentlig thrombocytæmi-lignende sygdom med øgede blodplader, øget antal megakaryocytter og knoglemarv fibrose. Tilsammen har disse modeller ikke kun gav mulighed for at få indsigt i den molekylære patogenese MPN, men også evnen til at udvikle og studere lægemidler i en præ-kliniske omgivelser.
Dette håndskrift indeholder en detaljeret beskrivelse af transduktion-transplantation metode med fokus på CALRdel52 mutationen. Denne teknik indebærer transplantation af retroviralt transducerede knoglemarvsceller, der udtrykker mutant konstrukt i bestrålede syngene recipientmus.
Protocol
Representative Results
Discussion
Denne protokol giver en detaljeret beskrivelse af, hvordan man udfører knoglemarvstransplantationer i mus til at rekapitulere en væsentlig thrombocythemi-lignende sygdom med progression til myelofibrose med CALRdel52 mutation som føreren af sygdom. Vellykket transplantation af celler, der udtrykker CALRdel52 resultater i øget blodplader, udvidelse af megakaryocytter og knoglemarvsfibrose. Som knoglemarvstransplantation er en flertrinsproces, er det vigtigt at erkende trin, hvor der kan undgås teknisk fejl at …
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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