Neuroprotocols最新文献

The ability to generate expanded populations of individual cell types able to undergo normal differentiation in vitro and in vivo is of critical importance in the investigation of the mechanisms that underlie differentiation and in studies on the use of cell transplantation to repair damaged tissues. This review discusses two different approaches to the generation of expanded cell populations with phenotypes useful for either of these purposes. In one line of research, an analysis of the growth control properties of glial precursor cells of the CNS has revealed that cooperation between appropriate mitogens can promote extended precursor division in the absence of differentiation, thus allowing unprecedented expansion of a primary cell population without resort to the expression of activated oncogenes in the cells of interest. In a second line of research, H-2K^btsA58 transgenic mice have been developed in order to allow the direct derivation of conditionally immortal cell lines from many tissues of the body simply by dissection and growth of cells under permissive conditions. In both instances, cells grown for extended periods in vitro displayed normal patterns of differentiation when reintroduced in vivo. In addition, conditionally immortal astrocytes derived from H-2K^btsA58 mice appear to offer a simple cellular model for studying the ability of glial scar tissue to inhibit migration of glial precursor cells and extension of neurites from mature neurons.

Three clonal gonadotropin-releasing hormone (GnRH) neuronal cell lines were derived from a genetically induced tumor in transgenic mice. A transgene was constructed to target expression of simian virus 40 T antigen to GnRH neurons using the promoter/enhancer domains of the cell-specifically expressed GnRH gene. The resulting GT1 cells were characterized by morphology, the expression of neuron-specific genes, expression and processing of GnRH, pulsatile basal secretion of GnRH, release of GnRH in response to depolarization, and regulation of GnRH release by a variety of neurotransmitters and neuromodulators. By all of these criteria, GT1 cells are highly differentiated neuronal cell lines that provide valuable models for studying the cell biology of neuroendocrine cells.

A useful approach to molecular biologic studies of complex mammalian systems is the judicious use of clonal cell lines. For studies on neuronal populations of the central nervous system, useful cell lines are lacking. A general protocol that produces clonal lines by retroviral infection in vivo followed by in vitro infection with an additional oncogene-containing virus is detailed. The lines produced express several neuronal markers but not glial fibrillary acidic protein. The lines are phenotypically stable, freezable, and transfectable by standard methods. Retroviral combinations that have produced stable lines using this protocol are large T with v-src and v-myc, and large T with v-ras. Other oncogenic combinations may produce different, useful phenotypes for studies on the molecular development and function of CNS neurons.

We describe the isolation and characterization of six immortal cell lines derived from primary cultures of olfactory epithelium (OE) purified from E15 mouse embryos. Cultured cells were immortalized using a replication-defective murine retrovirus containing the cDNA for human c-myc, in addition to a dominant selectable antibiotic resistance gene (neo). Cells that survived antibiotic selection and displayed process-bearing morphologies were expanded and analyzed for expression of molecular markers characteristic of olfactory receptor neurons, sustentacular cells, and olfactory ensheathing cells. Interestingly, all six cell lines expressed morphological and immunological properties of the ensheathing, or Schwann, cells of the olfactory nerve, but did not express markers characteristic of olfactory receptor neurons. Our results suggest that, while it is possible to generate immortalized OE cell lines using retrovirus-mediated oncogene transfer, there may be limitations to the types of cells that can be immortalized. In addition, we demonstrate the potential usefulness of immortalized OE cell lines for promoter-trap experiments to identify developmentally regulated genes.