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. 1978 Mar;75(3):1190–1193. doi: 10.1073/pnas.75.3.1190

Replica plating and in situ enzymatic assay of animal cell colonies established on filter paper.

J D Esko, C R Raetz
PMCID: PMC411435  PMID: 349559

Abstract

We have developed a simple technique for the replica plating of Chinese hamster ovary (CHO) cells. In this procedure cells are allowed to divide for 8-16 days between the plastic surface of a petri dish and a disc of Whatman no. 50 filter paper, weighted down with glass beads. The culture medium can be replaced when necessary without disturbing the growing colonies. Cells from each developing colony grow into the fibers of the paper, while others remain attached to the plate. The cell colonies transferred to the paper are viable and can be replica plated to a new petri dish with high resolution. In this way several inositol auxotrophs have been identified in a stock of mutagen-treated cells without prior enrichment. Alternatively, the cells on the paper can be rendered permeable in situ, which permits autoradiographic screening for specific biochemical defects, as reported previously for Escherichia coli [Raetz, C. R.H. (1975 Proc. Natl. Acad. Sci. USA 72, 2274-2278]. This technique is applicable to other common cell lines and is especially useful for the identification of single colonies defective in the synthesis of DNA, RNA, protein, and membrane lipids.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  2. Chang T. Y., Telakowski C., Heuvel W. V., Alberts A. W., Vagelos P. R. Isolation and partial characterization of a cholesterol-requiring mutant of Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1977 Mar;74(3):832–836. doi: 10.1073/pnas.74.3.832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang T. Y., Vagelos P. R. Isolation and characterization of an unsaturated fatty acid-requiring mutant of cultured mammalian cells. Proc Natl Acad Sci U S A. 1976 Jan;73(1):24–28. doi: 10.1073/pnas.73.1.24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DULBECCO R., VOGT M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med. 1954 Feb;99(2):167–182. doi: 10.1084/jem.99.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. HAM R. G. CLONAL GROWTH OF MAMMALIAN CELLS IN A CHEMICALLY DEFINED, SYNTHETIC MEDIUM. Proc Natl Acad Sci U S A. 1965 Feb;53:288–293. doi: 10.1073/pnas.53.2.288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kao F. T., Puck T. T. Genetics of somatic mammalian cells, VII. Induction and isolation of nutritional mutants in Chinese hamster cells. Proc Natl Acad Sci U S A. 1968 Aug;60(4):1275–1281. doi: 10.1073/pnas.60.4.1275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kuroki T. Agar plate cultrue and Lederberg-style replica plating of mammalian cells. Methods Cell Biol. 1975;9(0):157–178. [PubMed] [Google Scholar]
  8. LEDERBERG J., LEDERBERG E. M. Replica plating and indirect selection of bacterial mutants. J Bacteriol. 1952 Mar;63(3):399–406. doi: 10.1128/jb.63.3.399-406.1952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Raetz C. R., Chu M. Y., Srivastava S., Turcotte J. G. A phospholipid derivative of cytosine arabinoside and its conversion to phosphatidylinositol by animal tissue. Science. 1977 Apr 15;196(4287):303–305. doi: 10.1126/science.191910. [DOI] [PubMed] [Google Scholar]
  10. Raetz C. R. Isolation of Escherichia coli mutants defective in enzymes of membrane lipid synthesis. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2274–2278. doi: 10.1073/pnas.72.6.2274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Raetz C. R. Phosphatidylserine synthetase mutants of Escherichia coli. Genetic mapping and membrane phospholipid composition. J Biol Chem. 1976 Jun 10;251(11):3242–3249. [PubMed] [Google Scholar]
  12. Rao R. H., Strickland K. P. On the solubility, stability and partial purification of CDPdiacyl-sn-glycerol: inositol transferase from rat brain. Biochim Biophys Acta. 1974 May 29;348(2):306–314. doi: 10.1016/0005-2760(74)90243-4. [DOI] [PubMed] [Google Scholar]
  13. Saito Y., Chou S. M., Silbert D. F. Animal cell mutants defective in sterol metabolism: a specific selection procedure and partial characterization of defects. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3730–3734. doi: 10.1073/pnas.74.9.3730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Stamato T. D., Hohmann L. K. A replica plating method for CHO cells using nylon cloth. Cytogenet Cell Genet. 1975;15(6):372–379. doi: 10.1159/000130536. [DOI] [PubMed] [Google Scholar]
  15. Stamato T. D., Waldren C. A. Isolation of UV-sensitive variants of CHO-K1 by nylon cloth replica plating. Somatic Cell Genet. 1977 Jul;3(4):431–440. doi: 10.1007/BF01542971. [DOI] [PubMed] [Google Scholar]
  16. Villarreal L. P., Berg P. Hybridization in situ of SV40 plaques: detection of recombinant SV40 virus carrying specific sequences of nonviral DNA. Science. 1977 Apr 8;196(4286):183–185. doi: 10.1126/science.191907. [DOI] [PubMed] [Google Scholar]

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