Gavrilescu L Cristina, Van Etten Richard A
Molecular Oncology Research Institute, Tufts-NEMC, USA.
J Vis Exp. 2007(10):550. doi: 10.3791/550. Epub 2007 Dec 4.
Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFRalpha, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80 degrees C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.
我们的实验室研究由诸如Bcr-Abl等致癌基因或生长因子受体衍生的致癌基因（ZNF198-FGFR1、Bcr-PDGFRα等）诱导的人类骨髓增殖性疾病。通过移植先前感染了表达感兴趣致癌基因的逆转录病毒的骨髓细胞，我们能够在小鼠模型中模拟和研究类似人类的疾病。编码人类致癌基因和标记物（绿色荧光蛋白、红色荧光蛋白、抗生素抗性基因等）的复制缺陷型逆转录病毒是通过使用293T细胞的瞬时转染方案产生的，293T细胞是一种由腺病毒E1A基因产物转化的人肾上皮细胞系。293细胞具有通过磷酸钙（CaPO4）进行高效转染的特殊性质，很容易达到高达50%-80%的转染效率。在这里，我们将表达感兴趣致癌基因的逆转录病毒载体与表达gag-pol-env包装功能的质粒（如单基因组包装构建体kat或pCL，在这种情况下为EcoPak质粒）共转染293细胞。通过使用氯喹进一步提高初始转染效率。转染后48小时收集的嗜亲性病毒原液作为培养上清液，可以储存在-80℃，用于转化和体外研究中感染细胞系，或用于感染原代细胞，如小鼠骨髓细胞，然后可用于我们的小鼠模型移植。
