年月 | 事項 | 文献・参考資料 |
1951年 | HeLa細胞樹立 (George Gey) . | (1) |
1968年 | アイソザイム分析法によりHeLa細胞のクロスコンタミを指摘(Gartler、HeLaコンタミ)。賛否両論で混乱。 | (2,3) |
1978年 | HeLaコンタミに結論(ATCCが全収集ヒト細胞を調査:HeLaマーカー染色体の確認) | (4) |
1985年 | Jeffreys によるDNAフィンガープリント法の開発により、ヒト細胞相互の識別を可能になった。多数の細胞を比較することが困難だった(サザンハイブリダイゼーションとRIの使用がルーチン化の妨げになった)。 | (5,6) |
1991年 | Edwards らによるSTR分析法の開発が進む(PCR based STR analysis)。 | (8) |
1993年 | JCRB細胞バンクで、VNTRとサザンブロット法によりクロスコンタミの調査を行いMolt 4, P39など数種類の誤同定された細胞を発見し、よりシステマチックな細胞検査法が必要であることを痛感。この時期の方法ではRIを使用して扱いが煩雑だったうえに、電気泳動の結果を標準化し難いことが弱点だった。 | (7,12,13,14) |
1998年頃 | 日本組織培養学会・細胞バンク委員会では、理研細胞バンク(大野)、東北大細胞バンク(工藤)、JCRB細胞バンク(水澤)がそれぞれにクロスコンタミネーションの検出を試み結果を持ち寄って検討した。試みたのは酵素反応法(アメロゲニン検査、大野)、STR分析法(工藤)、VNTR分析法(水澤)の3法。 | - |
1999年 | 1998年頃の検討結果から工藤が調べていたSTR法が優れていると感じたので、すぐにJCRB細胞バンクでもこの方法を取り入れて分析を開始した。この方法が優れていたのは、実験が標準化されており結果を「データベース」化することが容易だったことにある。この検索システムはすぐにホームページ上に公開した。同じ頃ATCCでも同法による調査を開始した (プロメガ PowerPlex 1.2)。 | (7,9) |
2000年 | 米SIVB学会を通じて複数の細胞バンクがクロスコンタミ問題を憂慮していることが報告された。 | . |
2002年 | 英Mastersが呼びかけ世界中に分散しているHeLa細胞を集めて(200以上)STR分析を実施。他の細胞から区別されることを確認し、STR分析法の妥当性が示された。日本からはJCRB細胞バンクと理研細胞バンクが参加。 | (10) |
2007年 4月 | Science誌が Mistaken Identity として掲載。。 | (11) |
2007年 7月 | 米細胞生物学会がクロスコンタミネーションをニュースレターに掲載。 | (下記資料) |
2007年 7月 | Nardoneら、米DHHSのセクレタリー Leavett にオープンレターを送付。 | . |
2007年11月 | JCRB細胞バンクに Nardone から警告のメッセージが着信。 | (下記資料) |
2007年11月 | BBCラジオで40分のドキュメンタリー("Cancer research wasted millions")を放送。
| (BBC News) |
2008年 3月 | 細胞の誤同定の問題についてのアクション、Dr.Nardone からのメール(pdf)。
| . |
Nelson-Rees は培養細胞を使った研究から誤同定の問題の深刻さに気付き取組んできました。大変参考になります。(Nelson-Reesの論文)。 |
文献
Gey,G.O., Coffman,W.D., and Kubicek,M.T. Tissue culture studies of the proliferative capacity of cervical carcinoma and normal epithelium, Cancer Res., 12: 264-265, 1952.
Gartler,S.M. Genetic markers as tracers in cell culture, Natl. Cancer Inst. Monogr., 26: 167-195, 1967.
Notes: Same as the refer ID# 383.This is the first paper representing the tissue culture cross-contamination of HeLa cells. This paper deals with the use of various levels of genic expression to distinguish between cell cultures of different origin. DNA replication patterns, variations in the types of RNA molecules synthesized by different tissues, and the persistence of certain genetic markers in cell culture are considered. It is suggested that DNA-RNA hybridization as a means of comparing tissue and cell culture RNA molecules offers a promising tool for answers to questions of tissue origin-cell culture relationships. A survey of certain genetic markers in established human cell lines revealed the possibility of a serious problem of intraspecific contamination. Eighteen established human cell lines of supposed independent origin were shown to be identical with regard to their glucose-6-phosphate dehydrogenase (G6PD) (A) and phosphoglucomutase (1) types.
Reference ID: 1811
Gartler,S.M. Apparent HeLa cell contamination of human heteroploid cell lines, Nature, 217: 750-751, 1968.
Notes: Interspecific cell culture contamination has been detected several times by karyotypic and immunological procedures. These same measurements are of little value as detectors of intraspecific contamination, but polymorphic variants detectable at the cell culture level can be very useful for this purpose.
Reference ID: 365
Lavappa,K.S. Survey of ATCC stocks of human cell lines for HeLa contamination, In Vitro Cell Dev. Biol., 14: 469-475, 1978.
Notes: Seed stocks of human cell lines deposited at the American Type Culture Collection (ATCC) have been examined for cross-contamination with HeLa cells using Giemsa banded marker chromosomes. Sixteen additional cell lines investigated have been found to exhibit marker chromosomes typical of HeLa cells. Quinacrine fluorescence studies further revealed the absence of Y chromosome in these lines. These observations indicate that the lines are HeLa derivatives.
Reference ID: 371
Gill,P., Jeffreys,A.J., and Werrett,D.J. Forensic application of DNA 'fingerprints', Nature, 318: 577-579, 1985.
Jeffreys,A.J., Wilson,V., and Thein,S.L. Hypervariable minisatellite regions in human DNA., Nature, 314: 67-73, 1985.
Notes: The human genome contains many dispersed tandem-repetitive 'minisatellite' regions detected via a shared 10-15-base pair 'core' sequence similar to the generalized recombination signal (x) of Escherichia coli.
Reference ID: 86
Tanabe,H., Takada,Y., Minegishi,D., Kurematsu,M., Masui,T., and Mizusawa,H. Cell line individualization by STR multiplex system in the cell bank found cross contamination between ECV304 and EJ-1/T24., Tiss. Cult. Res. Commun., 18: 329-338, 1999.
Notes: Short tandem repeat (STR) regions represent highly polymorphic micro satellite markers in the human genome that have tandemly repetitive sequence elements of 2 to 7 bp in length as a unit The application of STR regions to population genetics and personal identification has been well studied. Recent technical advances have enabled us to analyze multilocus STR regions simultaneously by a method, called the STR Multiplex system, that uses a single PCR amplification in one tube. We established a new evaluation system for the identification of cell lines based on an STR Multiplex method that uses 9 different loci: D5S81 8. D13S31 7, D7S820, D16S539, vWA. THOI , Amelogenin, TPOX, and CSFIPO The STR profiling data from 96 cell lines were examined and an efficiency of this approach for cell standardization was found. Using this method, we have analyzed the STR profiles of human cell lines. ECV304, EJ-1, and T24, reoently reported by the DSMZ-German Collection of Microorganisms and Cell Cultures to have been cross-contaminated. Our results clearly detect the cross-contamination between ECV304 and EJ-1/T24. The cross-contamination was estimated to be derived from the T24 cells. Collectively, the STR Multipiex system provides a rapid, precise, and powerful method in cell line identification for quality control at the JCRB Cell Bank.
Reference ID: 5018
Edwards,A., Civitello,A., Hammond,H.A., and Caskey,C.T. DNA typing and genetic mapping with trimeric and tetrameric tandem repeats, Am. J. Hum. Genet., 49: 746-756, 1991.
Notes: Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX 77030 ENGLISH Tandemly reiterated sequences represent a rich source of highly polymorphic markers for genetic linkage, mapping, and personal identification. Human trimeric and tetrameric short tandem repeats (STRs) were studied for informativeness, frequency, distribution, and suitability for DNA typing and genetic mapping. The STRs were highly polymorphic and inherited stably. A STR- based multiplex PCR for personal identification is described. It features fluorescent detection of amplified products on sequencing gels, specific allele identification, simultaneous detection of independent loci, and internal size standards. Variation in allele frequencies were explored for four U.S. populations. The three STR loci (chromosomes 4, 11, and X) used in the fluorescent multiplex PCR have a combined average individualization potential of 1/500 individuals. STR loci appear common, being found every 300-500 kb on the X chromosome. The combined frequency of polymorphic trimeric and tetrameric STRs could be as high as 1 locus/20 kb. The markers should be useful for genetic mapping, as they are sequence based, and can be multiplexed with the PCR. A method enabling rapid localization of STRs and determination of their flanking DNA sequences was developed, thus simplifying the identification of polymorphic STR loci. The ease by which STRs may be identified, as well as their genetic and physical mapping utility, give them the properties of useful sequence tagged sites (STSs) for the human genome initiative
Reference ID: 4982
Mizusawa,H., Tanabe,H., Takada,Y., Kurematsu,M., Hojoh,M., Yasuda,R., and Masui,T. Cross culture contamination of cultured cell lines in Japan: JCRB case report and the use of DNA profiling for the sample control frame, In Vitro Cell Dev. Biol., 40: 10-A, 2004.
Notes: A-23
Reference ID: 5527
Masters,J.R., Thomson,J.A., Daly-Burns,B., Reid,Y.A., Dirks,W.G., Packer,P., Toji,L.H., Ohno,T., Tanabe,H., Arlett,C.F., Kelland,L.R., Harrison,M., Virmani,A., Ward,T.H., Ayres,K.L., and Debenham,P.G. Short tandem repeat profiling provides an international reference standard for human cell lines, Proc. Natl. Acad. Sci. U. S. A, 98: 8012-8017, 2001.
Notes: Institute of Urology, University College London, 3rd Floor Research Laboratories, 67 Riding House Street, London W1W 7EY, United Kingdom JMasters@Uclacuk
Abstract: Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated
Reference ID: 5216
Chatterjee,R. Cases of mistaken identity, Science., 315: 928-931, 2007.
Notes: For decades, biologists working with contaminated or misidentified cell lines have wasted time and money and produced spurious results; journals and fundin agencies say it's not their job to solve this problem. In the 1980s, When he was a postdoctoral fellow at the Scripps Research Institute in San Diego, Caligornia, Reinhard Kofler received what was supposed to be a human cancer cell line from a collaborator. "We cultured it, wecloned genes into it," he recalls, then "[we]genotayped it and realized it was 100% mouse."
Reference ID: 6120
Honma, M., Stacey, G., and Mizusawa, H., DNA profiling with polymorphic DNA markers, 9A:5.1-9A:5.13. Cell & Tissue Culture: Laboratory Procedures, John Wiley & Sons, Chichester, 1993.
Note:Very details of the experimental procedures for DNA profiling are described.
RefID: 3704
Honma,M., Kataoka,E., Ohnishi,K., Kikuno,T., Hayashi,M., Sofuni,T., and Mizusawa,H. Detection of recombinational mutations in cultured human cells by Southern blot analysis with minisatellite DNA probes, Mutat. Res., 286: 165-172, 1993.
Notes: Division of Genetics and Mutagenesis, National Institute of Hygienic Sciences, Tokyo, Japan 0165-1110 ENGLISH NETHERLANDS Using the human acute monocytic leukemia cell line, THP-1, a hypermutability of minisatellite loci was demonstrated in cell culture by Southern blot analysis with minisatellite DNA probes. DNA was isolated from 98 subclones and hybridized to a panel of minisatellite probes consisting of three multilocus minisatellite probes (ML probes) and seven locus-specific minisatellite probes (LS probes). The Southern blot patterns of the hybridized subclones were compared with those of the parental THP-1. Four mutated bands with two ML probes and two mutated bands with two LS probes were detected. The mutation frequency was estimated roughly at 0.1% based on the total number of bands analyzed, and it was much higher than that expected for other DNA regions. Four of these mutations were thought to be alterations of repetitions caused by insertion or deletion of tandem repeats, and one mutant lost a complete minisatellite allele. The nature of the sixth mutant was unclear. Because of the hypermutability of minisatellite DNA, Southern blot analysis using minisatellite DNA probes can be used as a mutation assay system directly based on the DNA JOURNAL-ARTICLE 0; 0; 0 93211420 9307
Reference ID: 4717
Honma,M., Mitani,K., Mizusawa,H., Hayashi,M., Sofuni,T., Muramatsu,M., and Komonami,R. A new VNTR polymorphism at locus D1S340, Hum. Mol. Genet., 2: 1329, 1993.
Notes: Division of Genetics and Mutagenesis, National Institute of Hygienic Sciences, Tokyo, Japan 0964-6906 ENGLISH ENGLAND JOURNAL-ARTICLE EC 3.1.21.-; EC 3.1.21.4; 0 94004892 9401
Reference ID: 4713
解説及び資料