ВИДОВАЯ ИДЕНТИФИКАЦИЯ КЛЕТОЧНЫХ КУЛЬТУР: РАЗРАБОТКА НОВЫХ ПОДХОДОВ ОСНОВАННЫХ НА ПОЛИМЕРАЗНОЙ ЦЕПНОЙ РЕАКЦИИ С ДЕТЕКЦИЕЙ ПРОДУКТА В РЕАЛЬНОМ ВРЕМЕНИ (ПЦР-РВ)
Abstract
Testing of species specificity is a major requirement to maintain the identity of cell lines during long-term
culturing. We developed a new method of identification of cell lines from the following species: Ovis aries
(sheep), Bos taurus (cow) and Sus scrofa (pig) . The method is based on polymerase chain reaction with
real time detection of the amplification product. For species identification the sequences of mitochondrial
DNA encoding cytochrome b and cytochrome c oxidase were used. Specificity of PCR was achieved with
validated primers and probes. High sensitivity of the method allowed to reliably detect single cells of an irrelevant
species as an admixture to hundreds of thousands cells of the species whose identity is analyzed.
Eight cell lines and 17 sublines were tested, and their identity was confirmed. Thus, our method is advantageous
over conventional karyologic and isoenzyme analyses or traditional PCR-based, electrophoresis-assisted
identification of species.
culturing. We developed a new method of identification of cell lines from the following species: Ovis aries
(sheep), Bos taurus (cow) and Sus scrofa (pig) . The method is based on polymerase chain reaction with
real time detection of the amplification product. For species identification the sequences of mitochondrial
DNA encoding cytochrome b and cytochrome c oxidase were used. Specificity of PCR was achieved with
validated primers and probes. High sensitivity of the method allowed to reliably detect single cells of an irrelevant
species as an admixture to hundreds of thousands cells of the species whose identity is analyzed.
Eight cell lines and 17 sublines were tested, and their identity was confirmed. Thus, our method is advantageous
over conventional karyologic and isoenzyme analyses or traditional PCR-based, electrophoresis-assisted
identification of species.
About the Author
К. Кулешов
ВНИИ экспериментальной ветеринарии им. Я.Р. Коваленко РАСХН (ВИЭВ),
Москва (Россия) лаборатория клеточной биотехнологии
Russian Federation
Russian Federation
References
1. Дьяконов Л.П., Ситьков В.И. (ред.). Животная клетка в культуре. М.: Спутник+, 2000, 400 с., илл.
2. Altschul SF, Madden TL, Schaeffer AA, Zhang J, Zhang Z, Miller W, Lipma DJ (1997) Gapped BLAST and PSI BLAST:a new generation of protein database search programs. Nuc Acid Res 25:3389-3402
3. Clausen J.J. Comparative chromosomal study of 31 cultured mammalian cell lines [Text]/ Clausen J.J., Syverton J.T. // J Natl Cancer Inst. 1962. Vol. 28. P. 117-45.
4. Cooper J. Species identification in cell culture: a two-pronged molecular approach [Text]/ Cooper J.. [et al.] // In Vitro Cellular & Developmental Biology Animal. 2007. Vol. 43. № 10. P. 344-351.
5. Coriell L.L. Common antigens in tissue culture cell lines [Text]/ Coriell L.L., Tall M.G., Gaskill H. // Science. 1958. Vol. 128. № 3317. P. 198-9.
6. Defendi V. Immunological and karyological criteria for identification of cell lines [Text]/ Defendi V., Billingham R.E., Silvers W.K., Moorhead P. // J Natl Cancer Inst. 1960. Vol. 25. P. 359-85.
7. Gartler S.M. Apparent Hela cell contamination of human heteroploid cell lines [Text]/ Gartler S.M. // Nature. 1968. Vol. 217. № 5130. P. 750-1.
8. Hay R.J. Methods for authenticating cell lines [Text]/ Hay R.J. // Dev Biol Stand. 1992. Vol. 76. -. - P. 25-37.
9. Liu M. Rapid identification and authentication of closely related animal cell culture by polymerase chain reaction [Text]/ Liu M., Liu H., Tang X., Vafai A. // In Vitro Cell Dev Biol Anim. 2008. Vol. 44. № 7. P. 224-227.
10. Losi C. An alternative method to isoenzyme profile for cell line identification and interspecies crosscontaminations: cytochrome b PCR-RLFP analysis [Text]/ Losi C., Ferrari S., Sossi E., Villa R., Ferrari M. // In Vitro Cellular & Developmental Biology - Animal. 2008.
11. Milanesi E. Molecular detection of cell line crosscontaminations using amplified fragment length polymorphism DNA fingerprinting technology [Text]/ Milanesi E.. [et al.] // In Vitro Cell Dev Biol Anim. 2003.Vol. 39. № 3-4. P. 124-30.
12. Ono K. Species identification of animal cells by nested PCR targeted to mitochondrial DNA [Text]/ Ono K.. [et al.] // In Vitro Cell Dev Biol Anim. 2007. Vol. 43. № 5-6. P. 168-75.
13. Parodi B. Species identification and confirmation of human and animal cell lines: a PCR-based method [Text]/ Parodi B.. [et al.] // Biotechniques. 2002. Vol. 32. № 2. P. 432-4, 436, 438-40.
14. Robin E.D. Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells [Text]/ Robin E.D., Wong R. // J Cell Physiol. 1988. Vol. 136. № 3. P. 507-13.
15. Simpson W.F. Species Identification of Animal Cell Strains by Immunofluorescence [Text]/ Simpson W.F., Stulberg C.S. // Nature. 1963. Vol. 199. P. 616-7.
16. Stacey G.N. DNA fingerprinting transforms the art of cell authentication [Text]/ Stacey G.N., Bolton B.J., Doyle A. // Nature. 1992. Vol. 357. № 6375. P. 261-2.
17. Steube K. Identification and verification of rodent cell lines by polymerase chain reaction [Text]/ Steube K., Koelz A.-L., Drexler H. // Cytotehnology. 2008. Vol. 56. № 1. P.49-56.
18. Steube K.G. A simple method using beta-globin polymerase chain reaction for the species identification of animal cell lines-a progress report [Text]/ Steube K.G., Meyer C., Uphoff C.C., Drexler H.G. // In Vitro Cell Dev Biol Anim. 2003. Vol. 39. № 10. P. 468-75.
19. Tsareva A.A.Isoenzyme characteristics of glucose- 6-phosphage dehydrogenase and lactate dehydrogenase of continuous cells from the collection of the Sverdlovsk Research Institute of Virus Infections [Text]/ Tsareva A.A., Glinskikh N.P., Dreizin R.S., Kolesnikova G.G., Zhdanov V.M. // Vopr Virusol. 1976. № 5. P. 620-3.
20. Molecular cloning : a laboratory manual / Sambrook, Joseph; Russell, David W. 3rd ed. New York: Cold Spring Harbor Laboratory, 2001.
Review
For citations:
. Russian Journal of Veterinary Pathology. 2008;(3):40-45. (In Russ.)
Views:
64
Email this article 