Generation of microglial cell lines by transfection with simian virus 40 large T gene

Neuroscience Letters, 141 (1992) 139 142 *) 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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Generation of microglial cell lines by transfection with simian virus 40 large T gene Y o s h i t a k a H o s a k a ", A k i h i k o K i t a m o t ¢ "b, M a s a t o S h i m o j & b, K a z u y u k i N a k a j i m a ~, Yoshirori Imai ~', Hiroshi H a n d a ~ a n d Shinichi K o h s a k a ~' "Department olNeurochemistry. National Institute of Neuroscience, Tokyo (Japan), hF~/i Central Research Laboratory, Mochida Pharmaceutical Co., Ltd., Shizuoka (Japan) and 'Department q[' Biomolecular Engineering, Faculty q[' Bioscience and Biotechnoloj,,y, Tokyo blstitute o[' Technoh~y, Kanagawa (Japan j (Received 26 February 1992; Revised version received 6 April 1992: Accepted 7 April 1992)

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Microglia: SV40 large T antigen: Transfection: Cell culture: Cell line

Microglial cells, which were isolated from a primary culture of neonatal rat brain, were transfected with temperature-sensitive simian virus 40 (SV40) large T gene by the calcium phosphate precipitation method. Four weeks after transfection, several colonies were generated, and cloned cells were characterized. One of the cloned cells (RBM129) proliferated actively at 37°C and the dividing rate was significantly decreased at 40.5°C. The expression of large T antigen was detected by western blotting in cells incubated at both 37°C and 40.5°C. The cell line showed high activity of non-specific esterase, isolectin B4 binding and phagocytosis. Also the cells were stained by ED I monoclonal antibody. These results indicate that these cells were derived from rat brain microglia, and immortalized by large T gene.

In recent years, many functional roles of glial cells in the central nervous system have been discussed. Microglia, one kind of glial cells, have been considered to play important roles in normal brain development [5, 12] and in pathological states [13, 20]. Recently, microglia have been isolated from neonatal rat brain and cultured [5, 14, 19]. It has been demonstrated that microglia produce nerve growth factor [11], basic fibroblast growth factor [17] and several cytokines [3, 4, 6], and that they have the capacity to express class II major histocompatibility complex antigen [4]. We previously showed that microglial conditioned medium has neurotrophic activity on embryonic rat neocortical neurons [14]; but is seems to be difficult to purify or identify the neurotrophic substances fi'om microglial conditioned medium, because of the limited amount of the medium. To solve this problem, it is of advantage to use migroglial cell lines. In this study we show that in vitro transfection with SV40 large T antigen gene results in the generation of rat microglial cell lines. To construct a plasmid, the viral DNA of SV40 strain tsl00 [21] was completely digested with EcoRI and par('orre,s'pondence." S. Kohsaka, Department of Neurochemistry, National Institute of Neuroscience, 4-1-10gawahigashi, Kodaira. Tokyo 187, Japan. Fax: (81) 423-46-1751.

tially digested with Pvull. The DNA fragment containing the entire SV40 early region was inserted into the EcoRI PvuII sites of pBR322. To make plasmids which contained origin-defective mutants, the plasmid DNA was partially digested with Bg/I, treated with SI nuclease and ligated [7]. One of the plasmids which contained origin-defective mutants was selected. The resulting plasmid DNA was digested with EcoRl and PvuII, and ligated with XbaI linkers. After digestion with Xbal, the DNA fragment containing the SV40 early region was isolated. A plasmid, named pUCSVOD.tsT, was constructed by inserting the DNA fragment into the Xbal site of pUC19. Plasmid pSV2neo [2] was used for isolation of neomycin-resistant cells. A primary microglial cell culture was prepared from the neocortical tissue of neonatal embryonic rat brain as described previously [14, 15]. The primary cultured microglial cells were cotransfected with pUCSVOD.tsT and pSV2neo by the calcium phosphate precipitation method [8] at a 10:1 molar ratio. Two weeks after transfection, the cells were treated with 0.05% Trypsin, the detached cells were discarded and the strongly adhering cells were cultured for another 2 weeks in the medium containing 400 y g of G418 per ml. Four weeks after transfection, colony formation was detected, and several colonies were cloned by limiting dilution. Transformed cell cultures were maintained in a 100-

140 m m dish with Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal calf serum, 50 U of penicillin per ml, 100 # g of streptomycin per ml and 3.7 g of NaHCO3 per liter in a 10% CO2 atmosphere at 37°C. Following 20 passages, growth curves and doubling time were obtained by counting the cells after detaching the cells with 0.05% Trypsin. The expression of large T antigen in the transformed cells was detected by Western blotting [15] with anti-large T monoclonal antibody (PAb416, Oncogene Science, USA)[9]. Immunocytochemical staining of the cells with antibodies to ED l, glial fibrillary acidic protein (GFAP), neurofilament (NF) and galactocerebroside (GC) was performed by the method described previously [14]. Fluorescein isothiocyanate (FITC)-labeled isolectin B4 [18] was used for detection of terminal a-D galactose on the cell surface. Non-specific esterase activity was detected by the method of Li et al. [10]. To determine the phagocytic activity, the cells were incubated in culture medium containing 0.2% polybead fluorescent microsphere solution as previously described [14]. The transfection of microglial cells with SV40 large T gene led to the generation of 52 clones, which were designated RBM101 to 152. A m o n g these clones, the strongest proliferating activity was observed in R B M 129 cells: therefore this clone was selected for further characterization. When the RBM129 cells were cultured at 37°C in medium containing 200 # g of G418 per ml, the cells actively proliferated and the doubling time was about 16 h (Fig. 1). As the cells reached the confluent stage, contact inhibition could be clearly seen. Since the temperature-

sensitive T-antigen does not function at higher temperalures, proliferating activity of the Iransfected cells were examined for at 40.5°(?. As shown in Fig. I, when cells were cultured at 40.5°C the dividing activity was dramatically decreased, although the expression of large T antigen was detected by western blotting in cells incubated at both 37°C and 40.5°C (Fig. 2). These results mdicate that RBM129 cells were immortalized by the temperature-sensitive large T gene. We further characterized the transformed cells by immunocytochemical, histochemical and biochemical methods. As shown in Fig. 3A, B, RBM129 cells were immunocytochemically stained by anti-ED 1 antibody, which is one of the markers for microglial cell [14]. It was also revealed that isolectin B4 bound to R B M I 2 9 cells (Fig. 3C, D). Furthermore, histochemical staining indicated that most of the cells possess high non-specifc esterase activity (Fig. 3G). In contrast, the cells were not stained by anti-NF, anti-GFAP or anti-GC antibodies (data not shown) suggesting that the cells are not derived from neurons, astrocytes or oligodendrocytes. RBM129 cells were found to actively engulf the fluorescent latex beads into their cytoplasm (Fig. 3E, F). These results clearly show that RBM129 cells were derived from rat brain microglia. Recently immortalization of mouse microglial cells by several oncogenes (v-myc, v-rail, v-raj) was reported [1,

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Fig. 1. Growth curve of immortalized RBM 129 cells. Cells (1×105)were seeded in dishes (100-mmdiameter) and cultured at 37°C or 40.5°C. At the times indicated, the cells were detached from the dishes with trypsin and counted with a hemocytometer,o, 37°C: O, 40.5°C.

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Fig. 2. Western blotting for large T antigen. RBMI29 cells were seeded at 37°C and incubated for 3 days at 37°C (lane 1) or 40.5°C (lane 2). Cells were harvested and solubilized, and equivalent amounts of protein were separated on 10 20% SDS PAGE and transferred to a nitrocellulose membrane. It was immunostained with anti-large T monoclonal antibody (PAb416), peroxidase-conjugatedgoat anti-mouse immunoglobulin G and ECL blotting reagent (Amersham).

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Fig. 3. Immunocylochemical staining with ED I monoclonal antibody (B), and the corresponding phase-contrast photomicrograph (A). Fluorescence 0fthe bound F1T('-isolectin B4 (D) and the corresponding phase-contrast photomicrograph (C). Fluorescence of the phagocytosed FlTC-latex beads (F) and the corresponding phase-contrast photomicrograph (E). Non-specific esterase staining (G).

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16]. In the present study, microglial cells of neonatal rat brain were successfully transformed by a plasmid containing the temperature-sensitive SV40 large T gene. The growth rate of the transformed cells could be regulated by the incubation temperature. We previously showed that microglial conditioned medium clearly enhanced the survivability of neurons and the neurite extension of neocortical neurons [14]. Preliminary investigations revealed that the same neurotrophic effects occurred in the conditioned medium of RBM129 (data not shown). These results suggest that RBM129 cells are useful for investigating the physiological significance of microglia in the central nervous system including the isolation and purification of neurotrophic factor(s) derived from microglia, and also for studying the biological nature of the microglial cell itself. This work was supported by the Japan Health Science Foundation and the Special Coordination Fund for promoting science and technology in Japan. 1 Blasi, E., Bocchini, V., Mazzolla, R. and Bistoni, F., Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus, J. Neuroimmunol., 27 (1990) 229 237. 2 Canaani, D. and Berg, P., Regulated expression of human interferon fl~ gene after transduction into cultured mouse and rabbit cells, Proc. Natl. Acad. Sci. USA, 79 (1982) 5166-5170. 3 Frei, K., Malipiero, U.V., Leist, T.P., Zinkernagel, R.M., Schwab, M.E. and Fontana, A., On the cellular source and function of interleukin 6 produced in the central nervous system in viral diseases, Eur. J. Immunol., 19 (1989) 689-694. 4 Frei, K., Siepl, C., Groscurth, P., Bodmer, S., Schwerdel, C. and Fontana, A., Antigen presentation and tumor cytotoxicity by interferon-lg-treated microglial cells, Eur. J. Immunol., 17 (1987) 1271 1278. 5 Giulian, D. and Baker, T.J., Characterization of ameboid microglia isolated from developing mammalian brain, J. Neurosci., 6 (1986) 2163 2178. 6 Giulian, D., Baker, T.J., Shih, L.-C.N. and Lachman, L.B., Interleukin 1 of the central nervous system is produced by ameboid microglia, J. Exp. Med., 164 (1986) 594 604. 7 Gluzman, Y., Sambrook, J.F. and Frisque, R.J., Expression of early genes of origin-defective mutants of simian virus 40, Proc. Natl. Acad. Sci. USA, 77 (1980) 3898 3902.

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