Cellular Signalling Laboratory, Department of Biomedical Sciences, University of Bologna, 40126 Bologna, Italy.
Inositide Laboratory, School of Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, Highfield, Southampton SO17 1BJ, UK.
Cells. 2024 Nov 8;13(22):1849. doi: 10.3390/cells13221849.
Efficient gene transduction and cell viability are critical factors in genetic manipulation for research and therapeutic purposes. In this study, we explored the challenges associated with transducing the NB-4 cell line, a well-established model for acute promyelocytic leukemia (APL), using lentiviral vectors. While the initial transduction efficiency in NB-4 cells reached approximately 30%, we observed a significant decrease in cell viability, a phenomenon not observed in other acute leukemia cell lines such as THP-1 cells. We identified that this toxicity could be mitigated by purifying viral particles through ultracentrifugation or polyethylene glycol (PEG) precipitation, indicating that toxic substances, potentially secondary metabolites released by HEK293, could be responsible for the cell death. Nevertheless, cell selection by puromycin was still ineffective; crucially, we discovered that the human phosphoglycerate kinase (hPGK) promoter, commonly used in the PLKO1 vector, may become silenced in NB-4 cells, preventing effective selection with puromycin. By replacing the hPGK promoter with the elongation factor-1 alpha (EF1α) promoter, we successfully achieved high transduction efficiency and robust selection, demonstrating the potential for this modified vector system to facilitate genetic studies in APL models. These findings provide important insights into optimizing gene transduction protocols not only for NB-4 cells but also for other challenging cell lines, offering a refined approach for gene delivery and selection in cell models.
高效的基因转导和细胞活力是研究和治疗目的遗传操作的关键因素。在这项研究中，我们探讨了使用慢病毒载体转导 NB-4 细胞系（一种用于急性早幼粒细胞白血病（APL）的成熟模型）所面临的挑战。虽然 NB-4 细胞中的初始转导效率达到了约 30%，但我们观察到细胞活力显著下降，而在其他急性白血病细胞系（如 THP-1 细胞）中并未观察到这种现象。我们发现，通过超速离心或聚乙二醇（PEG）沉淀纯化病毒颗粒可以减轻这种毒性，表明有毒物质，可能是 HEK293 释放的次级代谢产物，可能是导致细胞死亡的原因。然而，通过嘌呤霉素进行细胞选择仍然无效；至关重要的是，我们发现，PLKO1 载体中常用的人磷酸甘油酸激酶（hPGK）启动子在 NB-4 细胞中可能会沉默，从而阻止嘌呤霉素的有效选择。通过用延伸因子-1 阿尔法（EF1α）启动子替换 hPGK 启动子，我们成功实现了高效的转导效率和稳健的选择，表明这种改良的载体系统在 APL 模型中的基因研究中具有潜力。这些发现为优化基因转导方案提供了重要的见解，不仅适用于 NB-4 细胞，也适用于其他具有挑战性的细胞系，为细胞模型中的基因传递和选择提供了一种改进的方法。
