Nguyen Thanh Kim, Morse Sarah J, Fleischman Angela G
Department of Medicine, Division of Hematology/Oncology, University of California, Irvine.
Department of Medicine, Division of Hematology/Oncology, University of California, Irvine;
J Vis Exp. 2016 Dec 22(118):54624. doi: 10.3791/54624.
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.
转导移植是在小鼠中模拟人类血液系统恶性肿瘤的一种快速有效的方法。该技术可使目的基因在造血细胞中表达，并可用于研究该基因在正常和/或恶性造血过程中的作用。本方案以骨髓增殖性肿瘤（MPN）中最近发现的钙网蛋白（CALR）突变为例，详细描述了如何进行转导移植。在本方案中，用编码突变型CALR的逆转录病毒转导来自5-氟尿嘧啶（5-FU）处理的供体小鼠的全骨髓细胞，并将其移植到经致死性照射的同基因宿主中。表达突变型CALR的供体细胞用绿色荧光蛋白（GFP）标记。移植的小鼠会出现MPN表型，包括外周血中血小板升高、骨髓中巨核细胞扩增和骨髓纤维化。我们提供了如何产生逆转录病毒、计算病毒滴度、转导全骨髓细胞以及移植到受照射受体小鼠中的详细步骤说明。
