User Ratings

Easy to Follow Achieved Expected Results Overall Rating Add your comments

Immortalizing Central Nervous System Cells

Scott R. Whittemore¹

¹University of Miami School of Medicine, Miami, Florida

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 3.7

DOI:

10.1002/0471142301.ns0307s00

Online Posting Date:

May, 2001

GO TO THE FULL TEXT:

PDF or HTML at Wiley Online Library

Are you the author of this protocol? Login or register and return to this page.

Abstract

This unit presents methods for isolating clonal, neural-derived cell lines. One approach for isolating such neural cell lines involves a replication-deficient retrovirus encoding a specific oncogene and a selectable marker which are used to infect dissociated CNS cells dissected at a developmental stage at which the cell population of interest has not undergone its terminal mitotic division. Also presented is a method for cloning by limiting dilution, which may be necessary to obtain a pure population of cells. Following growth under appropriate selection conditions, clones are isolated and tested for their ability to differentiate with the desired phenotypic properties. A method is also provided for coating tissue culture dishes, which is necessary for successful culture of CNS neurons.

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Unit Introduction
Strategic Planning
Basic Protocol 1: Infection and Isolation of Clonal CNS Cell Lines
Alternate Protocol: Cloning by Limiting Dilution
Basic Protocol 2: Determining Cellular Phenotype and Lineage Potential
Support Protocol: Preparation of Collagen/Polylysine/HIHS-Coated Cell Culture Dishes
Reagents and Solutions
Commentary
Bibliography
Figures
Tables

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Infection and Isolation of Clonal CNS Cell Lines

Materials

Pregnant rat or mouse
DMEM/F-12 (appendix 2A; made using DMEM formulation lacking glucose, L-glutamine, phenol red, sodium bicarbonate, and sodium pyruvate), with and without 5% FBS
0.4% trypan blue in 0.85% NaCl, sterile
100-mm cell culture dishes coated with collagen, poly-l-lysine, and HIHS (prepared fresh; see Support Protocol)
Titered stock of retrovirus bearing the desired construct, in DMEM/F-12/5% FBS
1 mg/ml polybrene (hexadimethrine bromide; Abbott Labs) in water (filter sterilized and stored in aliquots at –20°C)
Selection medium (see recipe)
0.05% trypsin/0.53 mM EDTA, sterile (trypsin/EDTA)
FBS (appendix 2A)
Dimethyl sulfoxide (DMSO)

15-ml conical tubes (Falcon)
Silanized, fire-polished Pasteur pipets with tip diameter reduced to ~0.5 mm, sterile
Cell scraper, sterile
Beckman GRP, IEC MP4R, or equivalent refrigerated centrifuge
Cloning rings, sterile
Vacuum grease, autoclaved
Hemostats, sterile
24-well plates
30- and 100-mm tissue culture dishes
Cryovials
Additional reagents and equipment for counting cells, assessing cell viability with a hemacytometer and trypan blue, and trypsinizing cells (see cpmb appendix 3F and appendix 1A in this manual) for Southern blotting (cpmb unit 2.9A & cpmb unit 2.10)

Alternate Protocol: Cloning by Limiting Dilution

Additional Materials (also see Basic Protocol 1)

Medium conditioned for 24 hr on a dish of the cells to be cloned during exponential growth phase
96-well cell culture plates

Support Protocol: Preparation of Collagen/Polylysine/HIHS-Coated Cell Culture Dishes

Materials

0.1 mg/ml type IV collagen in water
0.1 mg/ml high-molecular-weight poly-l-lysine (MW >300,000) in water
Heat-inactivated horse serum (HIHS)
100-mm cell culture dishes

Looking for materials? Suppliers Guide

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 3.7.1

Hypothetical pathway for neural cell differentiation.

View Image

Literature Cited

Literature Cited
	Baetge, E.E., Behringer, R.R., Messing, A., Brinster, R.L., and Palmiter, R.D. 1988. Transgenic mice express the human phenylethanolamine-N-methyltransferase gene in adrenal medulla and retina. Proc. Natl. Acad. Sci. U.S.A. 85:3648-3652.
	Bayer, S.A., Altman, J., Russo, R.J., and Zhang, X. 1993. Timetables of neurogenesis in the human brain based on experimentally determined patterns in the rat. Neurotoxicology 14:83-144.
	Bottenstein, J.E. and Sato, G.H. 1979. Growth of a rat neuroblastoma cell line in serum-free supplemented media. Proc. Natl. Acad. Sci. U.S.A. 76:514-517.
	Brewer, G.J. and Cotman, C.W. 1989. Survival and growth of hippocampal neurons in defined medium at low density: Advantages of a sandwich culture technique or low oxygen. Brain Res. 494:65-74.
	Byrd, P.J., Grand, R.J.A., and Gallimore, P.H. 1988. Differential transformation of primary human embryo retinal cells by adenovirus E1 regions and combinations of E1A+ras. Oncogene 2:477-484.
	Efrat, S., Teitelman, G., Anwar, M., Ruggiero, D., and Hanahan, D. 1988. Glucagon gene regulatory region directs oncoprotein expression to neurons and pancreatic cells. Neuron 1:605-613.
	Eves, E.M., Kwon, J., Downen, M., Tucker, M.S., Wainer, B.H., and Rosner, M.R. 1994. Conditional immortalization of neuronal cells from postmitotic cultures and adult CNS. Brain Res. 656:396-404.
	Gage, F.H., Ray, J., and Fisher, L.J. 1995. Isolation, characterization, and use of stem cells from the CNS. Annu. Rev. Neurosci. 18:159-192.
	Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., and Bujard, H. 1995. Transcriptional activation by tetracyclines in mammalian cells. Science 268:1766-1769.
	Hockfield, S. and McKay, R.D.G. 1985. Identification of major cell classes in the developing mammalian nervous system. J. Neurosci. 5:3310-3328.
	Hoshimaru, M., Ray, J., Sah, D.W.Y., and Gage, F.H. 1996. Differentiation of the immortalized adult neuronal precursor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene. Proc. Natl. Acad. Sci. U.S.A. 93:1518-1523.
	Hunter, T. 1991. Cooperation between oncogenes. Cell 64:249-270.
	Jat, P.S., Noble, M.D., Ataliotis, P., Tanaka, Y., Yannoutsos, N., Larsen, L., and Kioussis, D. 1991. Direct derivation of conditionally immortal cell lines from an H-2k^b-tsA58 transgenic mouse. Proc. Natl. Acad. Sci. U.S.A. 88:5096-5100.
	Kershaw, T.R., Rashid-Doubell, F., and Sinden, J.D. 1994. Immunocharacterization of H-2K^b-tsA58 transgenic mouse hippocampal neuroepithelial cells. Neuroreport 5:2197-2200.
	Kinsella, A.R., Fiszer-Maliszewska, L., Mitchell, E.L.D., Guo, Y., Fox, M., and Scott, D. 1990. Introduction of the activated N-ras oncogene into human fibroblasts by retroviral vector induces morphological transformation and tumorigenicity. Carcinogen 11:1803-1809.
	Largent, B.L., Sosnowski, R.G., and Reed, R.R. 1993. Directed expression of an oncogene to the olfactory neuronal lineage in transgenic mice. J. Neurosci. 13:300-312.
	Mandel, G. and McKinnon, D. 1993. Molecular basis of neural-specific gene expression. Annu. Rev. Neurosci. 164:323-345.
	Marshall, C.J. 1991. Tumor suppressor genes. Cell 64:313-326.
	Mellon, P.L., Windle, J.J., Goldsmith, P.C., Padula, C.A., Roberts, J.L., and Weiner, R.I. 1990. Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5:1-10.
	Memberg, S.P. and Hall, A.K. 1995. Dividing neuron precursors express neuron-specific tubulin. J. Neurobiol. 27:26-43.
	Miller, D.G., Adam, M.A., and Miller, A.D. 1990. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol. Cell. Biol. 10:4239-4242.
	Mullen, R.J., Buck, C.R., and Smith, A.R. 1992. NeuN, a neuronal specific nuclear protein in vertebrates. Development 116:201-211.
	No, D., Yao, T.P., and Evans, R.M. 1996. Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 93:3346-3351.
	Paulus, W., Baur, I., Boyce, F.M., Breakefield, X.O., and Reeves, S.A. 1996. Self-contained, tetracycline-regulated retroviral vector system for gene delivery to mammalian cells. J. Virol. 70:62-67.
	Raff, M.C. 1989. Glial cell diversification in the rat optic nerve. Science 243:1450-1455.
	Sah, D.W.Y., Ray, J., Richard, N., Leisten, J., and Gage, F.H. 1996. Conditional immortalization of human neuronal glial and multipotent CNS progenitor cells. Abstr. Am. Soc. Neurosci. 22:29.
	Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning, A Laboratory Manual, 2nd ed., pp. 9.31-9.58. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
	Suri, C., Fung, B.P., Tischler, A.S., and Chikaraishi, D.M. 1993. Catecholaminergic cell lines from the brain and adrenal glands of tyrosine hydroxylase-SV40 T antigen transgenic mice. J. Neurosci. 13:1280-1291.
	Tohyama, T., Lee, V.M.-Y., Rorke, L.B., and Trojanowski, J.Q. 1991. Molecular milestones that signal axonal maturation and the commitment of human spinal cord precursor cells to the neuronal or glial phenotype in development. J. Comp. Neurol. 310:285-299.
	Wang, Y., O'Malley, B.W. Jr., Tsai, S.Y., and O'Malley, B.W. 1994. A regulatory system for use in gene transfer. Proc. Natl. Acad. Sci. U.S.A. 91:8180-8184.
	Whittemore, S.R. and Snyder, E.Y. 1996. The physiological relevance and functional potential of central nervous system–derived cell lines. Mol. Neurobiol. 12:13-38.
Key References
	Cepko, C.L. 1996a. Preparation of a specific retrovirus producer cell line. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 9.10.1-9.10.13. John Wiley & Sons, New York.
	Cepko, C.L. 1996b. Large-scale preparation and concentration of retroviral stocks. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 9.12.1-9.12.6. John Wiley & Sons, New York.
These references provide detailed methods for the production of high-titer retroviral producer cell lines and the isolation of infective retrovirus.
	Gage et al., 1995. above.
	Whittemore and Snyder, 1996. See above.
These references review the development and properties of CNS-derived neural cell lines and their uses both in vitro and in vivo.
Internet Resources
	http://www-leland.stanford.edu/group/nolan
This web page provides information on how to obtain and use the NX retrovirus producer lines, which can be used with transient transfection to generate high-titer retroviral stocks.