Lentivirus-mediated bifunctional cell labeling for in vivo melanoma study.
Lentivirus-mediated bifunctional cell labeling for in vivo melanoma study.
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DOI：
10.1111/j.1755-148x.2009.00545.x
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发表时间：
2009-06
影响因子：
4.3
通讯作者：
Merlino G
中科院分区：
文献类型：
--
作者：
Day CP;Carter J;Bonomi C;Esposito D;Crise B;Ortiz-Conde B;Hollingshead M;Merlino G
慢病毒载体（LV）能够标记多种细胞类型，实现转基因的稳定表达。然而，对于体内研究，标记基因表达的持续时间变化很大。我们开发了一系列含有不同启动子的 LV，用于在小鼠细胞中表达报告基因。几种 LV 标记的小鼠黑色素瘤细胞的长期培养和集落形成表明，源自哺乳动物持家基因的启动子，尤其是编码 RNA 聚合酶 II (Pol2) 和铁蛋白 (FerH) 的启动子，为报告基因表达提供了最高的一致性。对于体内研究，原发性 B16BL6 小鼠黑色素瘤感染了 LV，其荧光素酶-GFP 融合基因 (Luc/GFP) 由 Pol2 或 FerH 启动子驱动。当移植到同系 C57BL/6 小鼠中时，Luc/GFP 标记的 B16BL6 小鼠黑色素瘤细胞可以通过体内生物发光成像进行监测，并且可以通过 FACS 从肿瘤中分离出 GFP 阳性细胞。在 B16BL6 连续传代小鼠中，Pol2-Luc/GFP 标记虽然活性较低，但比 FerH-Luc/GFP 标记更可持续。我们得出的结论是，Pol-2-Luc/GFP 标记允许在免疫活性小鼠黑色素瘤模型中进行长期体内监测和肿瘤细胞分离。在这项研究中，我们开发并鉴定了慢病毒载体，使标记的小鼠黑色素瘤细胞能够维持双功能荧光素酶-GFP标记基因的长期一致表达，甚至在具有完整免疫功能的同基因小鼠中也是如此。该细胞标记系统可用于构建免疫活性小鼠黑色素瘤模型，允许肿瘤监测和基于 FACS 的肿瘤细胞从组织中分离，极大地促进黑色素瘤的体内研究。
Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long-term culture and colony formation of several LV-labeled mouse melanoma cells showed that promoters derived from mammalian house-keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase-GFP fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP-labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP-positive cells can be isolated from the tumors by FACS. Pol2-Luc/GFP labeling, while lower in activity, was more sustainable than FerH-Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol-2-Luc/GFP labeling allows long-term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models. In this study we have developed and identified lentiviral vectors that allow labeled mouse melanoma cells to maintain long-term and consistent expression of a bifunctional luciferase-GFP marker gene, even in syngeneic mice with an intact immune function. This cell-labeling system can be used to build immunocompetent mouse melanoma models that permit both tumor monitoring and FACS-based tumor cell isolation from tissues, greatly facilitating the in vivo study of melanoma.