Establishment and characterization of adenoviral E1A immortalized cell lines derived from the rat suprachiasmatic nucleus

Primary cultured cells from the presumptive anlage of the rat suprachiasmat ic nucleus (SCN) were immortalized by infection with a retroviral vector en coding the adenovirus 12S E1A gene. After drug selection, the resulting neu ral cell lines (SCN1.4 and SCN2.2) displayed (a) extended growth potential without evidence of transformed or tumorigenic properties, (b) expression o f E1A protein within all cell nuclei, and (c) heterogeneous cell types in v arious stages of differentiation. A large proportion of the SCN1.4 and SCN2 .2 cells were characterized by gliallike morphologies, but showed limited e xpression of corresponding cell type-specific antigens, In addition, both l ines exhibited a stable population of cells with neuronlike characteristics . When treated so as to enhance differentiation, these cells mere often dis tinguished by fine, long processes and immunocytochemical expression of neu ronal markers and peptides found within SCN neurons in situ. Observations o n SCN neuropeptide immunostaining, content, release, and mRNA expression fo llowed a concordant pattern in which somatostatin and vasopressin cells wer e the most and least common peptidergic phenotypes in both lines, respectiv ely, Since these results indicate that constituents of E1A-immortalized lin es derived from the primordial SCN can differentiate into cells with phenot ypes resembling parental peptidergic neurons, it will be critical to explor e next whether these lines also retain the distinctive function of the SCN to generate circadian rhythms. Cloning of immortalized cell types could sub sequently yield useful tools for studying the development of SCN glial and peptidergic cell types and delineating their distinct roles in mammalian ci rcadian timekeeping, (C) 1999 John Wiley & Sons, Inc.