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.
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.
转导移植是小鼠恶性血液病建模的有用方法。该技术已用于研究骨髓恶性肿瘤追溯到了BCR-ABL1的异位表达可以忠实地概括小鼠1慢性粒细胞白血病首次证明特别有价值。这一技术随后促进JAK2 V617F和MPL W515K / L的广泛研究突变骨髓增殖性疾病(MPN)。
MPN是一组恶性血液病特征在于成熟骨髓细胞和骨髓纤维化的生产过剩。这些疾病通常由造血干细胞的克隆扩增已在任一JAK2,MPL,或CALR获得的体细胞突变产生的。转导移植JAK2 V617F和MPL W515K / L车型展览真性红细胞增多症和骨髓纤维化2的临床特点– 5 </ SUP>。最近,也已用转导移植方法6产生钙网蛋白突变MPN的小鼠模型。这些小鼠发展的原发性血小板增多症样疾病具有增加血小板,巨核细胞的数量增加,和骨髓纤维化。一起,这些模型不仅提供了机会来洞察MPN的分子发病机制,而且要开发和临床前设置研究治疗剂的能力。
这份手稿提供转导移植方法,重点对CALRdel52突变的详细描述。该技术包括表达该突变体构造成照射同基因受体小鼠逆转录病毒转导骨髓细胞移植。
这个协议提供了如何在小鼠进行骨髓移植,以概括与进展中原发性血小板增多症样疾病对骨髓纤维化与CALRdel52突变疾病的驾驶员的详细描述。表达增加血小板,巨核细胞的扩张和骨髓纤维化CALRdel52结果细胞的移植成功。骨髓移植是一个多步骤的过程,它确认,其中可避免技术误差,以防止表达感兴趣甚至小鼠死亡的基因的细胞的植入差的步骤是重要的。
以一个常常被忽视一?…
The authors have nothing to disclose.
This work is supported by the V Foundation Scholar (AGF) and the MPN Research Foundation (AGF).
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 |