1. Analysis of the Cys82Arg mutation in follicle-stimulating hormone beta (FSHβ) using a novel FSH expression vector 
Andrew D Clark, Lawrence C Layman, M.D. 
Fertility and Sterility 
Volume 79, Issue 2, Pages (February 2003)
DOI: /S (02)

2. FIGURE 1
Schematic diagram of the pαFSHβ expression plasmid. The pαFSHβ is a dual expression vector that constitutively expresses the α and β subunits of FSH. The α subunit is inserted using BglII and NotI restriction sites present in the cloning site of the vector and is under the control of the human elongation factor 1α (EF-1α) promoter. The β subunit is flanked by a HindIII and SalI site and is under the control of a human cytomegalovirus promoter (CMV). In addition, the pαFSHβ expression construct includes a Zeocin resistance cassette for cell selection and a pUC origin to allow transfection and replication in bacterial cells.
Clark. Cys82 Arg mutation in FSHβ. Fertil Steril 2003.
Fertility and Sterility  , DOI: ( /S (02) )

3. FIGURE 2
Bioassay and immunoassay of the Cys82Arg mutant. Biological activity of the Cys82Arg construct was compared to wild-type FSH transfected CHO cells (Wild type), CHO cells not transfected with any expression construct (Untrans.), as well as fresh media (Media). In addition, the ability of the wild-type and the mutant C82R to be detected by immunoassay was also determined. Values are expressed as the raw data of immunoactivity or bioactivity when compared to identical tests run with known concentrations of WHO 71/223 hFSH protein standard. Normalization of the raw data to total protein extracted gave similar results. Each sample bar represents the mean of three separate cell colonies with ±95% confidence intervals error bars. Values for Cys82Arg, Untrans, and media only gave undetectable levels, so no confidence intervals could be calculated.
Clark. Cys82 Arg mutation in FSHβ. Fertil Steril 2003.
Fertility and Sterility  , DOI: ( /S (02) )

4. FIGURE 3
Radioimmunoassay for FAS of the Cys82Arg mutant. Media from CHO cells transfected with the Cys82Arg mutant (Cys82Arg), from wild-type FSH transfected CHO cells (Wild Type), untransfected CHO cells (Untrans.), and fresh cell media (Media) were assayed for FAS. FAS was detected using a polyclonal antibody specific for the conformation of FAS, which is slightly altered in its unbound state. Data are expressed as the mean of the raw data from three separate cell colonies. Cell colonies for Cys82Arg and wild type include error bars with ±95% C.I. Untransfected cells and fresh culture media had no detectable FAS, so no confidence intervals could be calculated. (Statistical analysis to compare samples was done with raw values normalized to total protein extracted; only the raw data are presented in this graph.)
Clark. Cys82 Arg mutation in FSH. Fertil Steril 2003.
Fertility and Sterility  , DOI: ( /S (02) )