Isolation of rat Schwann cell lines: use of SV40 T antigen gene regulated by synthetic metallothionein promoters

Synthetic promoter elements from the mouse metallothionein-I promoter controlling the expression of SV40 T antigen have been tested for their efficacy in cloning rat Schwann cell lines that retained the characteristic properties of these cells and could be passed in culture indefinitely. The constructed promoters contained either four (MT4) or five (MT5) copies of a metal regulatory element 5' to the CAAT and TATA elements from the HSV-1 thymidine kinase gene. Characterization of these promoters in transient expression assays and transformation assays showed that both MT5 and MT4 were approximately 10-fold and 15-fold, respectively, weaker than the wild-type MT-I promoter in the presence of heavy metal inducer. However, in the absence of inducer, the basal activity of both MT5 and MT4 was barely detectable and much lower than that of MT-I. Schwann cells were transfected with plasmids containing the SV40 T antigen gene under the control of the different metallothionein promoters and cell lines were established from each. Only with the MT5 and MT4 promoters were lines obtained that resembled secondary Schwann cells in culture in their morphology, generation time, and demonstration of contact inhibition. In the presence of zinc, the expression of T antigen in the lines derived with MT5 and MT4 was about 10-fold lower than that derived with MT-I. On removal of the inducer this level was reduced, and in one cell line T antigen was undetectable. Concomitant with the reduction in T antigen expression there was an increased expression of Po, a protein specific to myelin-forming Schwann cells, and a decreased expression of glial fibrillary acidic protein, a protein expressed only in nonmyelin-forming Schwann cells. These cell lines, therefore, closely resemble untransfected Schwann cells in culture.     

DNA-protein interactions in the CCAAT box region of the murine lactate dehydrogenase C promoter

Electrophoretic Mobility (EMSA), using oligonucleotides containing CCAAT box sequences from the murine Idhc promoter show the presence of CCAAT binding proteins in nuclear extracts from liver and testis. In a liver extract, a single shifted band is seen. However, in the testis extract, two shifts are observed, one of which may be due to a testis specific isoform of CCAAT binding factor (CBF). Southwestern analysis with an oligonucleotide probe containing these sequences reveals the presence of a protein of approximately 120 kD in the testis extract. In the liver extract, a 70-kD protein binds the probe. An antibody against HeLa CBF causes a supershift in testis nuclear extract.