Development and application of a stable HeLa cell line capable of site-specific transgenesis using the Cre-lox system: establishment and application of a stable TNFRI knockdown cell line to cytotoxicity assay.

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.