Generation and Analysis of Normal and <i>shiverer </i>Temperature-Sensitive Immortalized Cell Lines Exhibiting Phenotypic Characteristics of Oligodendrocytes at Several Stages of Differentiation

Abstract

Normal glial cells immortalized at specific developmental stages would be useful tools with which to study glial cell differentiation in vitro. Similarly, immortalized glial cell lines derived from known neurological mutants with identified developmental, molecular genetic defects would also be useful for the in vitro examination of the effects of the mutation on glial cell function. In this report we describe the immortalization of 19 separate oligodendroglial cell lines, 10 derived from normal mice and 9 derived from the neurological mutant shiverer, which is missing a large segment of the myelin basic protein gene. Enriched oligodendrocyte cultures prepared at 7 days in vitro, a time when the majority of the cells were oligodendrocyte precursors undergoing transition into mature oligodendrocytes, were immortalized with pZIPSVtsA58, which carries a temperature-sensitive immortalizing oncogene. All of the immortalized cell lines grew rapidly at the permissive temperature of 34 degrees C and exhibited a dramatic decrease in growth rate at the nonpermissive temperature of 39 degrees C. The cell lines were characterized by immunocytochemistry and Northern blot analysis for a number of glial cell markers, and the phenotype of all the lines were consistent with their being in the oligodendroglial cell lineage. The phenotypes of the cell lines varied significantly with representatives of oligodendrocyte precursors, and immature and mature oligodendrocytes being present within the population of immortalized lines. All the cell lines appeared to be clonal based upon Southern blot analysis and all have been passaged at least 40 times with retention of stable phenotype.