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

Fumiyo Saito, Hirofumi Yokota, Yoshihisa Sudo, Yoshikuni Yakabe, Haruko Takeyama, Tadashi Matsunaga

研究成果: Article

5 引用 (Scopus)

抄録

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 TNFα. 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.

元の言語English
ページ(範囲)1077-1087
ページ数11
ジャーナルToxicology in Vitro
22
発行部数4
DOI
出版物ステータスPublished - 2008 6
外部発表Yes

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Gene Transfer Techniques
Cytotoxicity
HeLa Cells
Assays
Genes
Cells
Cell Line
RNA Interference
Xenobiotics
RNA
Green Fluorescent Proteins
Transgenes
Toxicology
Functional analysis
Recombinases
Tumor Necrosis Factor Receptors
Chromosomes
Chromosomes, Human, Pair 9
Gene Knockout Techniques
Cryopreservation

ASJC Scopus subject areas

  • Toxicology

これを引用

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title = "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",
abstract = "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 TNFα. 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.",
keywords = "Cytotoxicity assay, Knock-down, Site-specific transgenesis, Stable cell line, TNFα/TNFRI",
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T1 - Development and application of a stable HeLa cell line capable of site-specific transgenesis using the Cre-lox system

T2 - Establishment and application of a stable TNFRI knockdown cell line to cytotoxicity assay

AU - Saito, Fumiyo

AU - Yokota, Hirofumi

AU - Sudo, Yoshihisa

AU - Yakabe, Yoshikuni

AU - Takeyama, Haruko

AU - Matsunaga, Tadashi

PY - 2008/6

Y1 - 2008/6

N2 - 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 TNFα. 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.

AB - 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 TNFα. 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.

KW - Cytotoxicity assay

KW - Knock-down

KW - Site-specific transgenesis

KW - Stable cell line

KW - TNFα/TNFRI

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