Saito Fumiyo, Yokota Hirofumi, Sudo Yoshihisa, Yakabe Yoshikuni, Takeyama Haruko, Matsunaga Tadashi
Chemicals Assessment Center, Environmental Effect Research Section, Chemicals Evaluation and Research Institute, Japan.
Toxicol In Vitro. 2008 Jun;22(4):1077-87. doi: 10.1016/j.tiv.2008.01.019. Epub 2008 Feb 10.
Mammalian cell models for gene knock-out/knock-in experiments are important for functional analysis of genes and have a potential of useful tool for toxicological studies. However, uncontrolled insertion of transgenes has raised significant concerns over unwanted side effects. To address this issue, we established a stable HeLa55 cell line capable of site-specific transgenesis by means of Cre-mediated cassette exchange at a site on the long arm of human chromosome 9 containing no constitutive transcripts. We applied HeLa55 to transgenesis of the green fluorescent protein (GFP) gene based on recombinase-mediated cassette exchange. The transformants stably expressed GFP transgenes, even after cryopreservation, without compromising physiological properties. We produced an RNA interference (RNAi)-inducible knockdown stable cell line against human tumor necrosis factor (TNF) receptor I, and one cloned stable cell line (TNFRIKD cells) exhibited long-term gene silencing with significant reduction (ca. 85%) and markedly resisted cytotoxicity induced by TNFalpha. Furthermore, xenobiotics were exposed to stable TNFRIKD cells and different cytotoxicity was exhibited based on various toxicological properties. Thus, we showed the feasibility of RNAi-based stable knockdown cells for xenobiotics-induced cytotoxicity, and HeLa55 has wide application for the generation of stable knock-in and knock-down cells mediated by RNAi.
用于基因敲除/敲入实验的哺乳动物细胞模型对于基因功能分析很重要，并且有潜力成为毒理学研究的有用工具。然而，转基因的非受控插入引发了对不良副作用的重大担忧。为了解决这个问题，我们建立了一种稳定的HeLa55细胞系，该细胞系能够通过Cre介导的盒式交换在人类9号染色体长臂上一个不包含组成型转录本的位点进行位点特异性转基因。我们基于重组酶介导的盒式交换将HeLa55应用于绿色荧光蛋白（GFP）基因的转基因。即使在冷冻保存后，转化体也能稳定表达GFP转基因，且不影响其生理特性。我们构建了一种针对人类肿瘤坏死因子（TNF）受体I的RNA干扰（RNAi）诱导的稳定敲低细胞系，其中一个克隆的稳定细胞系（TNFRIKD细胞）表现出长期基因沉默，显著降低（约85%），并明显抵抗TNFα诱导的细胞毒性。此外，将外源性物质暴露于稳定的TNFRIKD细胞，基于各种毒理学特性表现出不同的细胞毒性。因此，我们证明了基于RNAi的稳定敲低细胞用于外源性物质诱导的细胞毒性研究的可行性，并且HeLa55在通过RNAi介导产生稳定的敲入和敲低细胞方面具有广泛应用。
