骨髄増殖性腫瘍の形質導入 - 移植マウスモデル
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
形質移植は、マウスにおける血液悪性腫瘍をモデル化するための有用な方法です。この技術は、バックBCR-ABL1の異所性発現が忠実にマウス1に慢性骨髄性白血病を再現できた最初の実証にまでさかのぼる骨髄性悪性疾患を研究するために特に有用となっています。この技術は、その後骨髄増殖性腫瘍（MPN）を変異させJAK2 V617FおよびMPL W515K / Lの広範な研究を促進してきました。
MPNは、成熟骨髄細胞と骨髄線維症の過剰産生を特徴とする血液悪性腫瘍のグループです。これらの疾患は、一般的にはJak2、MPL、またはCALRのいずれかで体細胞変異を獲得した造血幹細胞のクローン性増殖から生じます。形質移植JAK2 V617FおよびMPL W515K / Lモデルの展示真性多血と骨髄線維症2の臨床的特徴– 5 </ SUP>。最近、カルレティキュリン変異MPNのマウスモデルはまた、形質移植法6で生成されています。これらのマウスは、増加した血小板、巨核球数の増加、および骨髄線維症本態性血小板血症のような疾患を発症します。一緒に、これらのモデルはMPNの分子病態だけでなく、前臨床設定において治療薬を開発し、研究するための能力への洞察を得る機会を提供していません。
この原稿はCALRdel52突然変異を中心とした形質導入移植方法論の詳細な説明を提供します。この技術は、変異体が照射同系のレシピエントマウスに構築物を発現するレトロウイルスで形質導入した骨髄細胞の移植を含みます。
Protocol
Representative Results
Discussion
このプロトコルは、疾患のドライバーとしてCALRdel52変異を有する骨髄線維症への進行と本態性血小板血症のような疾患を再現するために、マウスに骨髄移植を実行する方法の詳細な説明を提供します。増加した血小板、巨核球の拡大、および骨髄線維症にCALRdel52結果を発現する細胞の移植の成功。骨髄移植は、多段階プロセスであるので、技術的なエラーは、目的の遺伝子、あるいはマウス?…
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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