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 is frequently used to monitor transfection efficiency in transient transfection experiments. In this paper, we analyzed the suitability of the GH reporter gene to monitor transfection efficiency in COS-7 cells of an expression vector carrying the cDNA for the normal and mutated human follicle-stimulating hormone receptor (FSHR). The FSHR cDNA was cloned in the pSG5 expression vector and mutagenized (Ala307-->Thr) by oligonucleotide-mediated, site-directed mutagenesis. The expression plasmid pXGH5, carrying the structural gene for human GH, was used to monitor transfection efficiency. Different concentrations of pXGH5 and pSG5 containing normal or mutated FSHR cDNA were transfected in COS-7 cells by lipofection. The results showed: 1) The expression of pXGH5 was constant within individual experiments, but only in culture wells cotransfected with the same type of FSHR construct. On the contrary, the GH values normalized by the cell densities changed consistently depending 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 construct was quite variable between experiments, without any relationship to the type of FSHR construct. These data show that the GH secretion is not a good parameter to monitor the transfection efficiency of the FSHR in pSG5 in COS-7 cells. Nor are other parameters such as semiquantitative mRNA determination or ligand binding to the transfected receptor ideal when mutations resulting in changes in receptor function are expected.
Simoni
M
M
Institute of Reproductive Medicine of the University, Münster, Germany.
Gromoll
J
J
D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000096:biosynthesis; D018076:DNA, Complementary / Q000235:genetics*; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D019382:Human Growth Hormone / Q000235:genetics*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis; D011962:Receptors, FSH / Q000235:genetics*; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D011962:Receptors, FSH; D019382:Human Growth Hormone; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP