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. 1987 Nov 1;105(5):2315–2325. doi: 10.1083/jcb.105.5.2315

Transfection of neonatal rat Schwann cells with SV-40 large T antigen gene under control of the metallothionein promoter

PMCID: PMC2114872  PMID: 2824529

Abstract

Secondary cultures of Schwann cells were transfected with a plasmid containing the SV-40 T antigen gene expressed under the control of the mouse metallothionein-I promoter. We used the calcium phosphate method for transfection and obtained a transfection efficiency of 0.01%. The colonies were cloned by limited dilution, and these cloned cell lines were carried in medium containing zinc chloride (100 microM). One cloned cell line, which has now been carried for 180 doublings, appears to have a transformed phenotype with a doubling time of 20 h. These cells express SV-40 T antigen while maintaining established Schwann cell properties (positive staining for 217c, Ran-2, A5E3, glial fibrillary acidic protein, presence of 2',3'-cyclic nucleotide phosphohydrolase [CNPase] activity, and the ability to synthesize sulfogalactosylceramide and mRNA for the myelin protein, P0). Removal of zinc chloride from the medium resulted in reduced expression of T antigen and a change in the appearance of the cells to a more bipolar shape, although they still did not exhibit contact inhibition and maintained a doubling time of 20 h. These cells now became Ran-2- negative and showed increases in CNPase activity and in their ability to synthesize sulfogalactosylceramide. The amount of P0 mRNA remained unchanged. Transfected Schwann cells, however, stopped dividing when they contacted either basal lamina or neurites and became bipolar in appearance. The Schwann cells in contact with the neurites then extended processes to wrap around bundles of neurites. Transfection with the SV-40 T antigen gene therefore provides a method for obtaining Schwann cell lines that continue to express properties associated with untransfected cells in culture and may be used to study axon-Schwann cell interaction.

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Selected References

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  1. Aguayo A. J., Epps J., Charron L., Bray G. M. Multipotentiality of Schwann cells in cross-anastomosed and grafted myelinated and unmyelinated nerves: quantitative microscopy and radioautography. Brain Res. 1976 Mar 5;104(1):1–20. doi: 10.1016/0006-8993(76)90643-0. [DOI] [PubMed] [Google Scholar]
  2. BLACK P. H., ROWE W. P. SV-40 INDUCED PROLIFERATION OF TISSUE CULTURE CELLS OF RABBIT, MOUSE, AND PORCINE ORIGIN. Proc Soc Exp Biol Med. 1963 Dec;114:721–727. doi: 10.3181/00379727-114-28780. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Bray G. M., Aguayo A. J. Quantitative ultrastructural studies of the axon Schwann cell abnormality in spinal nerve roots from dystrophic mice. J Neuropathol Exp Neurol. 1975 Nov;34(6):517–530. doi: 10.1097/00005072-197511000-00006. [DOI] [PubMed] [Google Scholar]
  5. Brinster R. L., Chen H. Y., Warren R., Sarthy A., Palmiter R. D. Regulation of metallothionein--thymidine kinase fusion plasmids injected into mouse eggs. Nature. 1982 Mar 4;296(5852):39–42. doi: 10.1038/296039a0. [DOI] [PubMed] [Google Scholar]
  6. Brockes J. P., Fields K. L., Raff M. C. A surface antigenic marker for rat Schwann cells. Nature. 1977 Mar 24;266(5600):364–366. doi: 10.1038/266364a0. [DOI] [PubMed] [Google Scholar]
  7. Brockes J. P., Fields K. L., Raff M. C. Studies on cultured rat Schwann cells. I. Establishment of purified populations from cultures of peripheral nerve. Brain Res. 1979 Apr 6;165(1):105–118. doi: 10.1016/0006-8993(79)90048-9. [DOI] [PubMed] [Google Scholar]
  8. Brockes J. P., Raff M. C., Nishiguchi D. J., Winter J. Studies on cultured rat Schwann cells. III. Assays for peripheral myelin proteins. J Neurocytol. 1980 Feb;9(1):67–77. doi: 10.1007/BF01205227. [DOI] [PubMed] [Google Scholar]
  9. Brugge J. S., Butel J. S. Role of simian virus 40 gene A function in maintenance of transformation. J Virol. 1975 Mar;15(3):619–635. doi: 10.1128/jvi.15.3.619-635.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bunge M. B., Williams A. K., Wood P. M. Neuron-Schwann cell interaction in basal lamina formation. Dev Biol. 1982 Aug;92(2):449–460. doi: 10.1016/0012-1606(82)90190-7. [DOI] [PubMed] [Google Scholar]
  11. Bunge R. P., Bunge M. B., Eldridge C. F. Linkage between axonal ensheathment and basal lamina production by Schwann cells. Annu Rev Neurosci. 1986;9:305–328. doi: 10.1146/annurev.ne.09.030186.001513. [DOI] [PubMed] [Google Scholar]
  12. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  13. Colbère-Garapin F., Horodniceanu F., Kourilsky P., Garapin A. C. A new dominant hybrid selective marker for higher eukaryotic cells. J Mol Biol. 1981 Jul 25;150(1):1–14. doi: 10.1016/0022-2836(81)90321-1. [DOI] [PubMed] [Google Scholar]
  14. Durnam D. M., Palmiter R. D. Transcriptional regulation of the mouse metallothionein-I gene by heavy metals. J Biol Chem. 1981 Jun 10;256(11):5712–5716. [PubMed] [Google Scholar]
  15. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  16. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  17. Fields K. L., Brockes J. P., Mirsky R., Wendon L. M. Cell surface markers for distinguishing different types of rat dorsal root ganglion cells in culture. Cell. 1978 May;14(1):43–51. doi: 10.1016/0092-8674(78)90299-4. [DOI] [PubMed] [Google Scholar]
  18. Fields K. L., Dammerman M. A monoclonal antibody equivalent to anti-rat neural antigen-1 as a marker for Schwann cells. Neuroscience. 1985 Jul;15(3):877–885. doi: 10.1016/0306-4522(85)90085-5. [DOI] [PubMed] [Google Scholar]
  19. Fields K. L., Yen S. H. A subset of Schwann cells in peripheral nerves contain a 50-kDa protein antigenically related to astrocyte intermediate filaments. J Neuroimmunol. 1985 Jun;8(4-6):311–330. doi: 10.1016/s0165-5728(85)80070-9. [DOI] [PubMed] [Google Scholar]
  20. Frost E., Williams J. Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology. 1978 Nov;91(1):39–50. doi: 10.1016/0042-6822(78)90353-7. [DOI] [PubMed] [Google Scholar]
  21. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  22. Houweling A., van den Elsen P. J., van der Eb A. J. Partial transformation of primary rat cells by the leftmost 4.5% fragment of adenovirus 5 DNA. Virology. 1980 Sep;105(2):537–550. doi: 10.1016/0042-6822(80)90054-9. [DOI] [PubMed] [Google Scholar]
  23. Jessen K. R., Mirsky R. Nonmyelin-forming Schwann cells coexpress surface proteins and intermediate filaments not found in myelin-forming cells: a study of Ran-2, A5E3 antigen and glial fibrillary acidic protein. J Neurocytol. 1984 Dec;13(6):923–934. doi: 10.1007/BF01148594. [DOI] [PubMed] [Google Scholar]
  24. Kimura G., Itagaki A. Initiation and maintenance of cell transformation by simian virus 40: a viral genetic property. Proc Natl Acad Sci U S A. 1975 Feb;72(2):673–677. doi: 10.1073/pnas.72.2.673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lane D. P., Crawford L. V. T antigen is bound to a host protein in SV40-transformed cells. Nature. 1979 Mar 15;278(5701):261–263. doi: 10.1038/278261a0. [DOI] [PubMed] [Google Scholar]
  26. Lemke G., Axel R. Isolation and sequence of a cDNA encoding the major structural protein of peripheral myelin. Cell. 1985 Mar;40(3):501–508. doi: 10.1016/0092-8674(85)90198-9. [DOI] [PubMed] [Google Scholar]
  27. Linzer D. I., Levine A. J. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell. 1979 May;17(1):43–52. doi: 10.1016/0092-8674(79)90293-9. [DOI] [PubMed] [Google Scholar]
  28. Madrid R. E., Jaros E., Cullen M. J., Bradley W. G. Genetically determined defect of Schwann cell basement membrane in dystrophic mouse. Nature. 1975 Sep 25;257(5524):319–321. doi: 10.1038/257319a0. [DOI] [PubMed] [Google Scholar]
  29. Martin R. G., Chou J. Y. Simian virus 40 functions required for the establishment and maintenance of malignant transformation. J Virol. 1975 Mar;15(3):599–612. doi: 10.1128/jvi.15.3.599-612.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mirsky R., Jessen K. R. A cell surface protein of astrocytes, Ran-2, distinguishes non-myelin-forming Schwann cells from myelin-forming Schwann cells. Dev Neurosci. 1983;6(6):304–316. doi: 10.1159/000112357. [DOI] [PubMed] [Google Scholar]
  31. Mirsky R., Winter J., Abney E. R., Pruss R. M., Gavrilovic J., Raff M. C. Myelin-specific proteins and glycolipids in rat Schwann cells and oligodendrocytes in culture. J Cell Biol. 1980 Mar;84(3):483–494. doi: 10.1083/jcb.84.3.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Miyazaki K., Okamura N., Kishimoto Y., Lee Y. C. Determination of gangliosides as 2,4-dinitrophenylhydrazides by high-performance liquid chromatography. Biochem J. 1986 May 1;235(3):755–761. doi: 10.1042/bj2350755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Moya F., Bunge M. B., Bunge R. P. Schwann cells proliferate but fail to differentiate in defined medium. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6902–6906. doi: 10.1073/pnas.77.11.6902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Needham L. K., Tennekoon G. I., McKhann G. M. Selective growth of rat Schwann cells in neuron- and serum-free primary culture. J Neurosci. 1987 Jan;7(1):1–9. doi: 10.1523/JNEUROSCI.07-01-00001.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Osborn M., Weber K. Simian virus 40 gene A function and maintenance of transformation. J Virol. 1975 Mar;15(3):636–644. doi: 10.1128/jvi.15.3.636-644.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Peng W. W., Bressler J. P., Tiffany-Castiglioni E., de Vellis J. Development of a monoclonal antibody against a tumor-associated antigen. Science. 1982 Feb 26;215(4536):1102–1104. doi: 10.1126/science.7063842. [DOI] [PubMed] [Google Scholar]
  37. Petit C. A., Gardes M., Feunteun J. Immortalization of rodent embryo fibroblasts by SV40 is maintained by the A gene. Virology. 1983 May;127(1):74–82. doi: 10.1016/0042-6822(83)90372-0. [DOI] [PubMed] [Google Scholar]
  38. Raff M. C., Abney E., Brockes J. P., Hornby-Smith A. Schwann cell growth factors. Cell. 1978 Nov;15(3):813–822. doi: 10.1016/0092-8674(78)90266-0. [DOI] [PubMed] [Google Scholar]
  39. Raff M. C., Fields K. L., Hakomori S. I., Mirsky R., Pruss R. M., Winter J. Cell-type-specific markers for distinguishing and studying neurons and the major classes of glial cells in culture. Brain Res. 1979 Oct 5;174(2):283–308. doi: 10.1016/0006-8993(79)90851-5. [DOI] [PubMed] [Google Scholar]
  40. Raff M. C., Hornby-Smith A., Brockes J. P. Cyclic AMP as a mitogenic signal for cultured rat Schwann cells. Nature. 1978 Jun 22;273(5664):672–673. doi: 10.1038/273672a0. [DOI] [PubMed] [Google Scholar]
  41. Ranscht B., Clapshaw P. A., Price J., Noble M., Seifert W. Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2709–2713. doi: 10.1073/pnas.79.8.2709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  43. Salzer J. L., Williams A. K., Glaser L., Bunge R. P. Studies of Schwann cell proliferation. II. Characterization of the stimulation and specificity of the response to a neurite membrane fraction. J Cell Biol. 1980 Mar;84(3):753–766. doi: 10.1083/jcb.84.3.753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sogin D. C. 2',3'-Cyclic NADP as a substrate for 2',3'-cyclic nucleotide 3'-phosphohydrolase. J Neurochem. 1976 Dec;27(6):1333–1337. doi: 10.1111/j.1471-4159.1976.tb02612.x. [DOI] [PubMed] [Google Scholar]
  45. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  46. Stuart G. W., Searle P. F., Chen H. Y., Brinster R. L., Palmiter R. D. A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7318–7322. doi: 10.1073/pnas.81.23.7318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Stuart G. W., Searle P. F., Palmiter R. D. Identification of multiple metal regulatory elements in mouse metallothionein-I promoter by assaying synthetic sequences. 1985 Oct 31-Nov 6Nature. 317(6040):828–831. doi: 10.1038/317828a0. [DOI] [PubMed] [Google Scholar]
  48. Tegtmeyer P. Function of simian virus 40 gene A in transforming infection. J Virol. 1975 Mar;15(3):613–618. doi: 10.1128/jvi.15.3.613-618.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tjian R., Fey G., Graessmann A. Biological activity of purified simian virus 40 T antigen proteins. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1279–1283. doi: 10.1073/pnas.75.3.1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Weinberg H. J., Spencer P. S. Studies on the control of myelinogenesis. I. Myelination of regenerating axons after entry into a foreign unmyelinated nerve. J Neurocytol. 1975 Aug;4(4):395–418. doi: 10.1007/BF01261372. [DOI] [PubMed] [Google Scholar]
  53. White B. A., Bancroft F. C. Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples. J Biol Chem. 1982 Aug 10;257(15):8569–8572. [PubMed] [Google Scholar]
  54. van der Eb A. J., Graham F. L. Assay of transforming activity of tumor virus DNA. Methods Enzymol. 1980;65(1):826–839. doi: 10.1016/s0076-6879(80)65077-0. [DOI] [PubMed] [Google Scholar]

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