Establishment of human airway epithelial cells with doxycycline-inducible cell growth and fluorescence reporters
Establishment of human airway epithelial cells with doxycycline-inducible cell growth and fluorescence reporters
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建立具有强力霉素诱导细胞生长和荧光报告基因的人气道上皮细胞
DOI：
10.1007/s10616-021-00477-0
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发表时间：
2021
期刊：
影响因子：
2.2
通讯作者：
Fukuda Tomokazu
中科院分区：
文献类型：
--
作者：
Orimoto Ai;Takahashi Kohei;Imai Masaki;Kiyono Tohru;Kawaoka Yoshihiro;Fukuda Tomokazu
我们先前报道了通过共表达R24 C突变型细胞周期蛋白依赖性激酶4（CDK 4 R24 C）、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，我们描述了一种新的多顺反子慢病毒载体表达增殖因子，CDK 4 R24 C和细胞周期蛋白D1沿着与增强型绿色荧光蛋白（EGFP）下的多西环素（Dox）依赖的反式激活因子（rtTA）和四环素反应元件（TRE）。通过将Dox诱导的慢病毒载体导入人气道上皮细胞，我们建立了一种新的人气道上皮细胞系Tet-on K4 D，该细胞系含有多顺反子Dox诱导的CDK 4 R24 C和Cyclin D1。我们发现Tet-on K4 D细胞的生长可以被Dox控制。此外，K4 D基因和rtTA基因的表达可以通过荧光成像独立地监测。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，我们建立的人气道上皮细胞可用于各种研究，如肺病理学和生物学基础的分化过程，以形成复杂的假复层多细胞层。
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 (CDK4R24C), 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, CDK4R24Cand 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 CDK4R24Cand 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.
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影响因子：
3.7
作者：
Orimoto, Ai;Kyakumoto, Seiko;Fukuda, Tomokazu
通讯作者：
Fukuda, Tomokazu
影响因子：
23.9
作者：
Brambrink, Tobias;Foreman, Ruth;Jaenisch, Rudolf
通讯作者：
Jaenisch, Rudolf
DOI：
10.1513/pats.200805-041hr
发表时间：
2008-09-15
期刊：
Proceedings of the American Thoracic Society
影响因子：
--
作者：
Crystal, Ronald G;Randell, Scott H;Sunday, Mary E
通讯作者：
Sunday, Mary E
DOI：
10.1165/ajrcmb.10.1.7507342
发表时间：
1994-01-01
影响因子：
6.4
作者：
COZENS, AL;YEZZI, MJ;GRUENERT, DC
通讯作者：
GRUENERT, DC