Isolation of a potential neural stem cell line from the internal capsule of an adult transgenic rat brain

A thermosensitive mutation of simian virus 40 large T antigen (LTA) gene, t he tsA58 gene, was cloned downstream of the 6-kbp neurofilament light chain promoter in pPOLYIII and injected into the pronucleus of fertilised oocyte s of Sprague-Dawley rats to develop a strain harbouring six copies of the t ransgene. Immunocytochemical staining of hemizygous adult tissues with anti bodies to the C-terminus of LTA showed that the inactive form of LTA was ex pressed only in the fibres of the internal capsule and in the choroid plexu s of the brain. Culturing the former region at 33 degrees C, the permissive temperature for LTA, yielded a cell line, NF2C, which produced active LTA and grew at 33 degrees C but which produced only inactive LTA and eventuall y died at the nonpermissive temperature of 39 degrees C. This clonal cell l ine was heterogeneous at 33 degrees C, producing the precursor neuronal cel l marker nestin and the glial-specific markers glial fibrillary acidic prot ein, vimentin and S100A1, as well as weakly producing the neuronal cell mar kers 68-kDa neurofilament protein (NF68) and microtubule-associated protein 2 (MAP2) in different subpopulations of cells. However, at 39 degrees C, t he cells produced dendritic, neuronal-like processes and elevated levels of NF68 and MAP2, as well as the neuronal markers synaptophysin, neurone-spec ific enolase, and low levels of tau, all determined by western blotting and immunofluorescent staining. Basic fibroblast growth factor enhanced the gr owth of the cells at 33 degrees C but also enhanced the formation of dendri tic neuronal-like processes at 39 degrees C. It is suggested that NF2C repr esents a potential stem cell line from adult brain that expresses precursor and glial cell markers at 33 degrees C but undergoes partial differentiati on to a neuronal cell phenotype at 39 degrees C.