Mechanism of breakdown of blood-brain barrier accompanied with viral encephalopathy

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We have tried to establish immortalized mouse brain endothelial cell lines and rat astrocytes cell line for the co-culture system of both cells. Primary cultured mouse brain endothelial cells were transfected with SV40 T-antigen expressing vector and selected by zeocin and blasticidin. Primary cultured rat astrocytes were transfected with same vectors and selected by some procedures. Three colonies from endothelial cells and 10 colonies from astrocytes were obtained, but in all of the immortalized cell lines, expression of SV40 T was not controlled by tetracycline. Thus Tet-repressor expressing vector was re-transfected to the cell lines and finally 3 astrocyte cell lines were obtained in which expression of SV40 T was controlled by tetracycline. Now we are investigating whether the expression of GFAP changes or not by on/off of SV40 T, and trying to establish co-culture system of endothelial cell lines and astrocyte cell lines.