We previously reported the successful establishment of multiple immortalized cell lines that preserved the original nature of the primary cells via co-expression of R24C mutant cyclin-dependent kinase 4 (CDK4^R24C), Cyclin D1, and telomerase reverse transcriptase (TERT). However, as these genes are kind of oncogenes, tools to control their expression levels are favorable. In this study, we describe a new polycistronic lentiviral vector expressing proliferation factors, CDK4^R24C and Cyclin D1 along with enhanced green fluorescence protein (EGFP) under the control of doxycycline (Dox)-dependent transactivator (rtTA) and tetracycline response element (TRE). By introducing the Dox-inducible lentiviral vector into human airway epithelial cells, we established a novel human airway epithelial cell line harboring polycistronic Dox-inducible CDK4^R24C and Cyclin D1, referred to as Tet-on K4D cells. We showed that the cell growth of Tet-on K4D cells could be controlled by Dox. Furthermore, expression of K4D genes and rtTA gene can be independently monitored by fluorescent imaging. Cultured airway epithelial cells are useful as a tool for studying the pathogenesis of lung disorders. Altogether, our established human airway epithelial cells could be used for a variety of studies such as lung pathology and biology underlying the differentiation process to form the complex pseudostratified multicellular layers.

我们之前报道了通过共表达 R24C 突变细胞周期蛋白依赖性激酶 4 (CDK4 ^R24C )、细胞周期蛋白 D1 和端粒酶逆转录酶 (TERT) 成功建立了多个永生化细胞系，这些细胞系保留了原代细胞的原始性质。然而，由于这些基因是一种癌基因，控制其表达水平的工具是有利的。在这项研究中，我们描述了一种表达增殖因子的新多顺反子慢病毒载体 CDK4 ^R24C和 Cyclin D1 以及在强力霉素 (Dox) 依赖性反式激活因子 (rtTA) 和四环素反应元件 (TRE) 控制下的增强型绿色荧光蛋白 (EGFP)。通过将 Dox 诱导型慢病毒载体引入人气道上皮细胞，我们建立了一种新型人气道上皮细胞系，该细胞系含有多顺反子 Dox 诱导型 CDK4 ^R24C和 Cyclin D1，称为 Tet-on K4D 细胞。我们表明，Tet-on K4D 细胞的细胞生长可以由 Dox 控制。此外，可以通过荧光成像独立监测 K4D 基因和 rtTA 基因的表达。培养的气道上皮细胞可用作研究肺部疾病发病机制的工具。总之，我们建立的人类气道上皮细胞可用于各种研究，例如肺病理学和分化过程的生物学，以形成复杂的假复层多细胞层。