Transfection ofPinealocytes with ICER Constructs

A partial cDNA of the ICER 5'-end (0.2 kb) was generated by RT-PCR from RNA isolated from rat pineal glands as described earlier (65). The two primers used (forward: 5'-TTT-TGG-ACT-GTG-GTA-CGG-CC-3'; reverse: 5'-GGG GAC TGT GCA GGCTTC CT-3') encompass the start site of translation of the ICER gene (63,45) and the ICER coding sequence upstream the first DNA binding domain. PCR fragments were size-selected, electroeluted, subcloned into the vector pBS SK- and sequenced before further use. Prior to transfection of pinealocytes, the ICER cDNA fragment was subcloned into the eukaryotic expression vector pRc/CMV (Invitrogen) in either antisense (pICERas) or sense (pICERs) orientation. As an additional control, cells were transfected with the vector alone (pRC/CMV). To monitor transfection efficiency within a given experiment, cells were cotransfected with 10^g of the vector pCH110 (Pharmacia) that carries the -galactosidase gene. Primary pinealocyte cultures were prepared, maintained as described above and transfected by the calcium phosphate co-precipitation technique (51). The culture medium was supplemented with 20 |rg of pRC/CMV-DNA, pICERs or pICERas, respectively, and 10|rg pCH110 for 5 h. Subsequently, cultures were washed and after a recovery period of 16 h stimulated with 10-6M NE, 10-6M ISO and l0-7M PE for 5 h, and melatonin was measured in the medium.

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