Expression of Myelin Protein Genes and Other Myelin Components in an Oligodendrocytic Cell Line Conditionally Immortalized with a Temperature‐Sensitive Retrovirus

Abstract

Abstract:We have conditionally immortalized oligodendl-ocytes 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 (C4 18) 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. I cells to be positive for the oligodendrocyte surface markers-galactocerebroside, A007, and AZB5. 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 m R N A s isolated from N20.1 cells were translatable and directed the synthesis of the 17-, 1 8 5 , and 2 1.5-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.

The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

“…Our goal here was to elucidate the NLS in this isoform. The conditionally-immortalized N19 oligodendroglial line was chosen as a relevant stage-specific cell line to examine the translocation of this MBP isoform in tissue culture [36]. This cell line stains positive for both the NG2 and A2B5 antigens, which are markers for oligodendroglial progenitor cells (OPCs), and lacks expression of classic MBP and proteolipid protein mRNAs [35].…”

“…The FuGene HD transfection reagent was purchased from Roche Diagnostics (Indianapolis, IN). The immortalized N19-OLG cell lines [34][35][36] were grown in DMEM high-glucose media supplemented with 10% FBS (fetal bovine serum) and 1% penicillin/streptomycin, and cultured in 10 cm plates at 34 °C/5% CO 2 . At 70-80% confluency (4-7 days), cells were detached using 0.25% trypsin for 5 min.…”

The transcription factor E2F-1 modulates TGF-β1 RNA expression in glial cells

“…High levels of the viral T-antigen were expressed in N20.1 cells only at permissive temperature (348C), but not at nonpermissive temperature (398C) (Verity et al, 1993). Under similar conditions, the Tk1 mutant, defective in its ability to bind pocket-domain proteins was unable to promulgate conditions for E2F-1 activity (data not shown).…”

“…In order to further examine the requirement for Tantigen in the E2F-1-mediated regulation of TGF-b1 expression, a glial cell line, N20.1, stably transformed with a temperature-sensitive SV40 T-antigen was utilized (Verity et al, 1993). High levels of the viral T-antigen were expressed in N20.1 cells only at permissive temperature (348C), but not at nonpermissive temperature (398C) (Verity et al, 1993).…”

Regulation of Cellular MicroRNA Expression in Oligodendrocytes during Acute and Persistent Coronavirus Infection

“…The N20.1 cell is a cell line clone derived from mouse primary cultures of oligodendrocyte conditionally immortalized by transformation with a temperature sensitive mutant of the simian virus-40 large T-antigen [26]. It was kindly provided by Dr. Anthony Compadre [University of California Los Angeles School of Medicine].…”