MONITORING THE TRANSFECTION EFFICIENCY OF THE HUMAN FOLLICLE-STIMULATING-HORMONE RECEPTOR CDNA IN COS-7 CELLS - EVALUATION OF THE GROWTH-HORMONE TRANSIENT GENE-EXPRESSION ASSAY SYSTEM

The human growth hormone (GH) transient gene expression assay system i s frequently used to monitor transfection efficiency in transient tran sfection experiments. in this paper, we analyzed the suitability of th e GH reporter gene to monitor transfection effciency in COS-7 cells of an expression vector carrying the cDNA for the normal and mutated hum an follicle-stimulating hormone receptor (FSHR). The FSHR cDNA was clo ned in the pSG5 expression vector and mutagenized (Ala(307)->Thr) by o ligonucleotide-mediated, site-directed mutagenesis. The expression pla smid pXGH5, carrying the structural gene for human GH, was used to mon itor transfection efficiency. Different concentrations of pXGH5 and pS G5 containing normal or mutated FSHR cDNA were transfected in COS-7 ce lls by lipofection. The results showed: 1) The expression of pXGH5 was constant within individual experiments, but only in culture wells cot ransfected with the same type of FSHR construct. On the contrary, the GH values normalized by the cell densities changed consistently depend ing on the type of FSHR construct. 2) The expression of the GH plasmid was influenced by type and concentration of the cotransfected plasmid . 3) The expression of pXGH5 cotransfected with the same FSHR construc t was quite variable between experiments, without any relationship to the type of FSHR construct. These data show that the GH secretion is n ot a good parameter to monitor the transfection efficiency of the FSHR in pSG5 in COS-7 cells. Nor are other parameters such as semiquantita tive mRNA determination or ligand binding to the transfected receptor ideal when mutations resulting in changes in receptor function are exp ected.