DI TNC1

Studies of the development and biochemistry of the central neurosystem

Established by transfecting cultures of primary astrocytes from diencephalon tissue of a 1 day old sprague-dawley rat with a DNA construct containing the oncogenic early region of SV40. Transcriptional control was affected using the human GFAP promoter (pGFA-SV-Tt) and the murine phosphoglycerate kinase promoter (pPGK-neo). Cloning was achieved using G418. The cells are phenotypically similar to type 1 astrocytes, including immunoreactivity to glial fibrillary acid protein (GFAP) and a β-alanine inhibitable high-affinity uptake mechanism for gamma amino butyric acid (GABA). α-2-macroglobulin production is similar to that found in primary astrocytes, but transferrin production is reduced. The cell line has been shown not to produce proenkephalin A, galactocerebroside or to express the 04 or A2B5 epitopes characteristic of type 2 astrocytes. Immunostaining has shown the SV40 T antigen to be present in over 90% of cells.

Rat Astrocyte transfected

DMEM + 4mM Glutamine + 1mM Sodium Pyruvate (NaP) + 10% Foetal Bovine Serum (FBS).

Split sub-confluent cultures (70-80%) at 1:2 to 1:6 using 0.25% trypsin or trypsin/EDTA; 5% CO₂; 37°C. Subculture before flasks become confluent or cells will detach in sheets reducing effectiveness of trypsin.

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