EXPRESSION OF MYELIN PROTEIN GENES AND OTHER MYELIN COMPONENTS IN AN OLIGODENDROCYTIC CELL-LINE CONDITIONALLY IMMORTALIZED WITH A TEMPERATURE-SENSITIVE RETROVIRUS
EXPRESSION OF MYELIN PROTEIN GENES AND OTHER MYELIN COMPONENTS IN AN OLIGODENDROCYTIC CELL-LINE CONDITIONALLY IMMORTALIZED WITH A TEMPERATURE-SENSITIVE RETROVIRUS
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DOI：
10.1111/j.1471-4159.1993.tb03188.x
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发表时间：
1993-02-01
影响因子：
4.7
通讯作者：
CAMPAGNONI, AT
中科院分区：
文献类型：
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作者：
VERITY, AN;BREDESEN, D;CAMPAGNONI, AT
我们通过使用逆转录病毒载体ZIPSVtsA 58，从正常和颤抖的小鼠原代脑培养物中分离出了条件永生化的少突胶质细胞。该载体编码永生化热不稳定性猿猴病毒40大T抗原（Tag），并允许通过赋予新霉素（G418）抗性进行克隆选择。我们分离了14个颤抖细胞系和10个正常细胞系，它们表达早期少突胶质细胞标记物2 '，3'-环核苷酸3 '-磷酸二酯酶mRNA。这些细胞系在允许温度（34 ℃）下连续生长，并显示Tag核免疫染色。在转移到非允许温度（39 ℃）时，细胞显示出快速停滞的细胞生长和Tag染色的丧失。从正常少突胶质细胞工程化的一个系（N20.1）也表达髓鞘碱性蛋白（MBP）和蛋白脂质蛋白（PLP）mRNA，这些基因通常由成熟的分化的少突胶质细胞表达。与维持在34 ℃的细胞相比，在39 ℃下生长>9天的N20.1细胞中没有观察到任何髓鞘特异性蛋白mRNA水平的差异。免疫细胞化学染色显示N20.1细胞对少突胶质细胞表面标志物半乳糖苷、A007和A2 B5呈阳性。然而，MBP和PLP多肽不能检测到蛋白质印迹或免疫细胞化学染色在允许或非允许的温度。无细胞蛋白质合成实验表明，从N20.1细胞中分离的MBP mRNA是可翻译的，并指导17-，18.5-和21.5-kDa MBP异构体的合成。聚合酶链反应分析PLP/DM 20基因剪接产物表明，在该细胞系中，DM 20 mRNA的表达高于PLP mRNA的表达。由于该细胞系表达MBP和PLP基因，因此它代表了“成熟”的少突胶质细胞，但这些基因的剪接模式表明它处于“成熟”的早期阶段。该细胞系现已传代>40次，表型和基因型均保持保真度。
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-degrees-C) and displayed Tag nuclear immunostaining. On shifting to nonpermissive temperatures (39-degrees-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-degrees-C for >9 days compared with cells maintained at 34-degrees-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.