Electroporation of rat pituitary (GH) cell lines: optimal parameters and effects on endogenous hormone production

An efficient electroporation procedure was established for the genetic transformation of two clonal strains of hormone producing rat pituitary cells (GH12C1 and GH3). We used the bacterial chloramphenicol acetyltransferase (CAT) gene as reporter gene to determine optimal conditions for electroporation. The conditions found to be optimal, measured as expression of the highest CAT activity, were 240-300 V and a DNA concentration of 30-60 micrograms/ml in sucrose buffer. Cell viability was then about 50 per cent. Maximum CAT activity was seen 24 hours after electroporation. The electroporation procedure, in the presence or absence of DNA, caused a transient decrease in endogenous growth hormone (GH) and prolactin (PRL) production.     

Intracellular mechanisms of gonadotropin-stimulated gene expression in granulosa cells.

Previous studies have shown that the gonadotropins follicle-stimulating hormone and luteinizing hormone stimulate proopiomelanocortin (POMC) promoter activity and mRNA levels in ovarian granulosa cells. The objective of these studies was to determine the role of cAMP-dependent protein kinases (pKA) in gonadotropin-stimulated gene expression. Primary cultures of rat granulosa cells were transfected with a gene construct consisting of the POMC promoter (-150 to +63; designated pOMC-CAT) fused to the chloramphenicol acetyltransferase (CAT) reporter gene either alone or cotransfected with an expression plasmid (designated mutant RI), which overexpresses a mutant form of the murine RI subunit incapable of binding cAMP and serving as an irreversible inhibitor of the catalytic subunit of pKA. Follicle-stimulating hormone or isoproterenol caused a significant stimulation of pOMC-CAT activity in transfected cells. Cotransfection of pOMC-CAT with mutant RI caused a significant inhibition of basal pOMC-CAT activity and abolished the gonadotropin stimulation. As a control, transfection of the SV-40 viral enhancer-promoter fused to CAT (pSV2-CAT) was unresponsive to follicle-stimulating hormone stimulation and cotransfection with mutant RI had no significant effect on pSV2-CAT activity. These studies suggest that gonadotropin regulation of the POMC promoter is mediated by pKA and that promoter activity is stringently controlled by pKA.