• Astrocyte;
  • Cell immortalization;
  • Glial cells;
  • Oligodendrocyte;
  • Shiverer mutation;
  • SV40 large T antigen


  1. Top of page
  2. Abstract

Abstract: We have conditionally immortalized oligodendrocytes isolated from normal and shiverer primary mouse brain cultures through the use of the retroviral vector ZIPSVtsA58. This vector encodes an immortalizing thermolabile simian virus 40 large T antigen (Tag) and allows for clonal selection by conferring neomycin (G418) resistance. We isolated 14 shiverer and 10 normal lines that expressed the early oligodendrocyte marker 2′,3′-cyclic nucleotide 3′-phosphodiesterase mRNA. These cell lines grew continuously at the permissive temperature (34°C) and displayed Tag nuclear immunostaining. On shifting to nonpermissive temperatures (39°C), the cells showed rapid arrested cell growth and loss of Tag staining. One line (N20.1) engineered from normal oligodendrocytes also expressed myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs, genes normally expressed by mature, differentiated oligodendrocytes. No differences in any of the myelin-specific protein mRNA levels were observed in N20.1 cells grown at 39°C for >9 days compared with cells maintained at 34°C. Immunocytochemical staining revealed N20.1 cells to be positive for the oligodendrocyte surface markers—galactocerebroside, A007, and A2B5. However, MBP and PLP polypeptides could not be detected by western blot or immunocytochemical staining at either the permissive or nonpermissive temperature. Cell-free protein synthesis experiments indicated that the MBP mRNAs isolated from N20.1 cells were translatable and directed the synthesis of the 17-, 18.5-, and 21.5-kDa MBP isoforms. Analysis of the PLP/DM20 gene splice products by polymerase chain reaction indicated that the expression of DM20 mRNA predominated over that of PLP mRNA in this cell line. Because the cell line expressed the MBP and PLP genes, it represents a “mature” oligodendrocyte, but the splicing patterns of these genes indicate that it is at an early stage of “maturation’. This cell line has now been passaged >40 times with fidelity of phenotype and genotype.

Abbreviations Used:

2′,3′-cyclic nucleotide 3′-phosphodiesterase




glial fibrillary acidic protein


myelin-associated glycoprotein


myelin basic protein


phosphate-buffered saline


polymerase chain reaction


proteolipid protein


sodium dodecyl sulfate


saline–sodium citrate


saline–sodium phosphate–EDTA


simian virus


simian virus 40 large T antigen


Tris-buffered saline


  1. Top of page
  2. Abstract
  • Allinquant B., D'Urso D., Almazan G., and Colman D. R. (1990) Transfection of transformed shiverer mouse glial cell lines. Dev. Neurosci. 12, 340348.
  • Allinquant B., Stugaitis S. M., D'Urso D., and Colman D. R. (1991) The ectopic expression of myelin basic protein isoforms in shiverer oligodendrocytes: implications for myelinogenesis. J. Cell Biol. 113, 393403.
  • Alliot F. and Pessac B. (1984) Astrocyte cell clones derived from established cultures of 8-day postnatal mouse cerebella. Brain Res. 306, 283291.
  • Amur-Umarjee S. G., Dasu R. G., and Campagnoni A. T. (1990a) Temporal expression of myelin-specific components in neonatal mouse brain cultures: evidence that 2′,3′-cyclic nucleotide 3′-phosphodiesterase appears prior to galactocerebroside. Dev. Neurosci. 12, 251262.
  • Amur-Umarjee S. G., Hall L., and Campagnoni A. T. (1990b) Spatial distribution of mRNAs for myelin proteins in primary cultures of mouse brain. Dev. Neurosci. 12, 263272.
  • Barbarese E., Barry C., Chou C. J., Goldstein D. J., Nakos G. A., Hyde-DeRuyscher R., Scheld K., and Carson J. H. (1988) Expression and localization of myelin basic protein in oligodendrocytes and transfected fibroblasts. J. Neurochem. 51, 17371745.
  • Bell G. I., Karam J. H., and Rutter W. J. (1981) Polymorphic DNA region adjacent to the 5′ end of the human insulin gene. Proc. Natl. Acad. Sci. USA 78, 57595763.
  • Benda P., Lightbody J., Sato G., Levine L., and Sweet W. (1968) Differentiated rat glial cell strain in tissue culture. Science 161, 370371.
  • Bernier L., Alvarez F., Norgard E. M., Raible D. W., Mentaberry A., Schembri J. G., Sabatini D. D., and Colman D. R. (1987) Molecular cloning of a 2′,3′-cyclic nucleotide 3′-phosphodiesterase: mRNAs with different 5′ ends encode the same set of proteins in nervous and lymphoid tissues. J. Neurosci. 7, 27032710.
  • Bong M., Chakrabarti A., Banik N., Hogan E. L., Kanoh M., Wiggens R. C., and Konat G. (1991) Differential regulation of myelin gene expression in SV40 T antigen-transfected rat glioma C6 cells. Metab. Brain Dis. 6, 717.
  • Bourre F. and Sarasin A. (1983) Targeted mutagenesis of SV40 DNA induced by UV light. Nature 305, 6870.
  • Butel J. S. and Jarvis D. L. (1986) The plasma-membrane-associated form of SV40 large tumor antigen: biochemical and biological properties. Biochim. Biophys. Acta 865, 171195.
  • Campagnoni A. T. (1988) Molecular biology of myelin proteins from the central nervous system. J. Neurochem. 51, 114.
  • Campagnoni A. T. and Macklin W. B. (1988) Cellular and molecular aspects of myelin protein gene expression. Mol. Neurobiol. 2, 4189.
  • Carroll R. B., Samad A., Mann A., Harper J., and Anderson C. W. (1988) RNA is covalently linked to SV40 large T antigen. Oncogene 2, 437444.
  • Chen G. L., Halligan N. L. N., Lue N. F., and Chen W. W. (1987) Biosynthesis of myelin-associated proteins in simian virus 40 (SV40)-transformed rat Schwann cell lines. Brain Res. 414, 3548.
  • Choi B. H. and Kim R. C. (1985) Expression of glial fibrillary acidic protein by immature oligodendroglia and its implications. J. Neuroimmunol. 8, 215235.
  • Chomczynski P. and Sacchi N. (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156159.
  • Chou J. Y. (1989) Differentiated mammalian cell lines immortalized by temperature-sensitive tumor viruses. Mol. Endocrinol. 3, 15111514.
  • Denhardt D. T. (1966) A membrane filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641646.
  • Dupouey P., Jacque C., Bourre J. M., Cesselin F., Privat A., and Baumann N. (1981) Immunochemical studies of myelin basic protein in shiverer mouse devoid of major dense line of myelin. Neurosci. Lett. 12, 113115.
  • Edmond J., Robbins R. A., Bergstrom J. D., Cole R. A., and de Vellis J. (1987) Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes and oligodendrocytes from developing brain in primary culture. J. Neurosci. Res. 18, 551561.
  • Feinberg A. P. and Vogelstein B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 613.
  • Fields K. L. and Yen S. H. (1985) A subset of Schwann cells in peripheral nerves contain a 50-kDa protein antigenically related to astrocyte intermediate filaments. J. Neuroimmunol. 8, 311330.
  • Frederiksen K., Jat P. S., Valtz N., Levy D., and McKay R. (1988) Immortalization of precursor cells from the mammalian CNS. Neuron 1, 439448.
  • Gardinier M. V. and Macklin W. B. (1988) Myelin proteolipid protein gene expression in jimpy and jimpymsd mice. J. Neurochem. 51, 360369.
  • Geller H. M. and Dubois-Dalcq M. (1988) Antigenic and functional characterization of a rat central nervous system-derived cell line immortalized by a retroviral vector. J. Cell Biol. 107, 19771986.
  • Hayes C., Kelly D., Murayama S., Komiyama A., Suzuki K., and Popko B. (1992) Expression of the neu oncogene under the transcriptional control of the myelin basic protein gene in transgenic mice: generation of transformed glial cells. J. Neurosci. Res. 32, 175187.
  • Jat P. S. and Sharp P. A. (1989) Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature. Mol. Cell. Biol. 9, 16721681.
  • Kennedy P. G., Watkins B. A., Thomas D. G., and Noble M. D. (1987) Antigenic expression by cells derived from human gliomas does not correlate with morphological classification. Neuropathol. Appl. Neurobiol. 13, 327347.
  • Kim S. U. (1990) Neurobiology of human oligodendrocytes in culture. J. Neurosci. Res. 27, 712728.
  • Knapp P. E., Bartlett W. P., and Skoff R. P. (1987) Cultured oligodendrocytes mimic in vivo phenotype characteristics: cell shape, expression of myelin-specific antigens, and membrane production. Dev. Biol. 120, 356365.
  • Knapp P. E., Skoff R. P., and Maki A. A. (1992) Properties of glial cells which divide during CNS development. (Abstr.) Trans. Am. Soc. Neurochem. 23, 91.
  • Korr H., Schultze B., and Maurer W. (1975) Autoradiographic investigations of glial proliferation in the brain of adult mice. II. Cycle time and mode proliferation of neuroglial and endothelial cells. J. Comp. Neurol. 160, 477490.
  • Kronquist K. E., Crandall B. E., Macklin W. B., and Campagnoni A. T. (1987) Expression of myelin proteins in the developing human spinal cord: cloning and sequencing of human proteolipid protein cDNA. J. Neurosci. Res. 18, 395401.
  • Lewis S. A., Balcarek J. M., Krek V., Shelanski M., and Cowan N. J. (1984) Sequence of a cDNA encoding mouse glial fibrillary acidic protein: structural conservation of intermediate filaments. Proc. Natl. Acad. Sci. USA 81, 27432746.
  • Ogawa H., Sato Y., Takeshita I., Tateishi T., and Kitamura K. (1985) Transient expression of glial fibrillary acidic protein in developing oligodendrocytes in vivo. Dev. Brain Res. 18, 133141.
  • Peden K. W. C., Charles C., Sanders L., and Tennekoon G. I. (1989) Isolation of rat Schwann cell lines: use of SV40 T antigen gene regulated by synthetic metallothionein promoters. Exp. Cell Res. 185, 6072.
  • Perreau J., Lilienbaum A., Vasseur M., and Paulin D. (1988) Nucleotide sequence of the human vimentin gene and regulation of its transcription in tissues and cultured cells. Gene 62, 716.
  • Puvion E., Duthu A., Harper F., Ehrhart J. C., Viron A., and May P. (1988) Intranuclear distribution of SV40 large T-antigen and transformation-related protein p53 in abortively infected cells. Exp. Cell Res. 177, 7389.
  • Redies C., Lendahl U., and McKay R. D. G. (1991) Differentiation and heterogeneity in T-antigen immortalized precursor cell lines from mouse cerebellum. J. Neurosci. Res. 30, 601615.
  • Reidl L. S., Campagnoni C. W., and Campagnoni A. T. (1981) Preparation and properties of an immunosorbent column specific for myelin basic protein. J. Neurochem. 37, 373380.
  • Renfranz P. J., Cunningham M. G., and McKay R. D. G. (1991) Region-specific differentiation of the hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain. Cell 66, 713729.
  • Roach A., Takahashi N., Pravtcheva D., Ruddle F., and Hood L. (1985) Chromosomal mapping of mouse myelin basic protein gene and structure and transcription of the partially deleted gene in shiverer mutant mice. Cell 42, 149155.
  • Roth H. J., Kronquist K., Pretorius P. J., Crandell B. F., and Campagnoni A. T. (1986) Isolation and characterization of a cDNA coding for a novel human 17.3K myelin basic protein (MBP) variant. J. Neurosci. Res. 16, 227238.
  • Salzer J. L., Holmes W. P., and Colman D. R. (1987) The amino acid sequence of the myelin-associated glycoproteins: homology to the immunoglobulin gene superfamily. J. Cell Biol. 104, 957965.
  • SambrookJ., FritschE. F., and ManiatisT., eds (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor , New York .
  • Schindler P., Luu B., Sorokine O., Trifilieff E., and Van Dorsselaer A. (1990) Developmental study of proteolipids in bovine brain: a novel proteolipid and DM-20 appear before proteolipid protein (PLP) during myelination. J. Neurochem. 55, 20792085.
  • Skoff R. P. and Knapp P. E. (1991) Division of astroblasts and oligodendroblasts in postnatal rodent brain: evidence for separate astrocyte and oligodendrocyte lineages. Glia 4, 165174.
  • Sorg B. A., Smith M. M., and Campagnoni A. T. (1987) Developmental expression of the myelin proteolipid protein and basic protein mRNAs in normal and dysmyelinating mutant mice. J. Neurochem. 49, 11461154.
  • Suzumura A., Bhat S., Eccleston P. A., Lisak R. P., and Silberberg D. H. (1984) The isolation and long-term culture of oligodendrocytes from newborn mouse brain. Brain Res. 324, 379383.
  • Tegtmeyer P. (1975) Function of simian virus 40 gene A in transforming infection. J. Virol. 15, 613618.
  • Tennekoon G. I., Yoshino J., Peden K. W. C., Bigbee J., Rutkowski J. L., Kishimoto Y., DeVries G. H., and McKhann G. M. (1987) Transfection of neonatal rat Schwann cells with SV40 large T antigen gene under control of the metallothionein promoter. J. Cell Biol. 105, 23152325.
  • Timsit S. G., Bally-Cuif L., Colman D. R., and Zalc B. (1992) DM-20 mRNA is expressed during the embryonic development of the nervous system of the mouse. J. Neurochem. 58, 11721175.
  • Tooze J. (1981) DNA Tumor Viruses: Molecular Biology of Tumor Viruses, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor , New York .
  • Trapp B. D., Moench T., Pulley M., Barbosa E., Tennekoon G., and Griffen J. (1987) Spatial segregation of mRNA encoding myelin-specific proteins. Proc. Natl. Acad. Sci. USA 84, 77737777.
  • Trapp B. D., Small J. A., Pulley M., Khoury G., and Scangos G. A. (1988) Dysmyelination in transgenic mice containing JC virus early region. Ann. Neurol. 23, 3848.
  • Verdi J. M., Kampf K., and Campagnoni A. T. (1989) Translational regulation of myelin protein synthesis by steroids. J. Neurochem. 52, 321324.
  • Verity A. N. and Campagnoni A. T. (1988a) Regional expression of myelin protein genes in the developing mouse brain: in situ hybridization studies. J. Neurosci. Res. 21, 238248.
  • Verity A. N. and Campagnoni A. T. (1988b) Myelin basic protein gene expression in the quaking mouse. Brain Dysfunct. 1, 272284.
  • Westphal M., Brunken M., Rohde E., and Herrmann H. D. (1988) Growth factors in cultured human glioma cells: differential effects of FGF, EGF, and PDGF. Cancer Lett. 38, 283296.
  • Whittemore S. R., Holets V. R., Keane R. W., Levy D. J., and McKay R. D. G. (1991) Transplantation of a temperature-sensitive, nerve growth factor-secreting, neuroblastoma cell line into adult rats with fimbria–fornix lesions rescues cholinergic septal neurons. J. Neurosci. Res. 28, 156170.
  • Wight P. A., Verity A. N., Campagnoni A. T., and Macklin W. B. (1992) Transcriptional regulatory elements of the myelin proteolipid gene. (Abstr.) Trans. Am. Soc. Neurochem. 23, 210.