Core B: FSH Glycoform Production, Purification, and Analysis

项目总结/文摘:

Project Summary/Abstract: Core B – FSH Glycoform Production, Purification and Analysis Core B provides critical and requisite reagents, analyses, and expertise to support all of the Projects in fulfilling their Specific Aims. Specifically, Core B will continue to produce recombinant hFSH glycoforms and mutant hFSH glycoforms. Procedures have been established to produce milligram quantities of glycoform variants from the conditioned medium of cultured GH3 cells transfected with the human FSH a and b subunit genes, CGA and FSHB, respectively, or mutant FSHB genes. Core B will continue to develop transfection systems to improve mutant hFSH glycoform production to supply hormone to all four Projects. These hFSH variants mimic known recombinant hypoglycosylated hFSH glycoforms in terms of their actions upon ovarian cells. Core B will continue to work with Project 4(Bousfield), to generate and assess antibodies that recognize specifically intact hFSH, hFSH a subunit, as well as individual glycoform b subunits, for use in glycoform isolation and detection. Core B will provide a variety of cells for use in Project 2(Davis), Project 3(Jonas), and Project 4(Bousfield) to assess glycoform receptor binding, FSH receptor oligomerization, FSH receptor mediated signal transduction and glycoform impact upon cell differentiation. Cells to be furnished include KGN cells, a human granulosa cell line that responds to hFSH stimulation; CHO cells that express the human FSH receptor; and primary porcine granulosa cells which naturally express the FSH receptor to complete on-going experiments. GH3 and HEK293 cells transfected with hFSH and mutated genes are critical for the production of recombinant hFSH glycoforms. Core B will provide analysis support for the Projects including: FSH RIA and ELISA to monitor hFSH levels during FSH glycoform production and in various biological fluids including serum and urine; mouse FSH ELISA for Project 1(Kumar); FSH and LH receptor binding; progesterone ELISA to monitor hFSH variant impact upon target cell function; and hFSH variant antigen ELISA for monoclonal antibody screening. Core B will obtain and develop studies utilizing porcine cumulus oocyte complexes (pCOCs) to assess potential differential activities of hFSH glycoform variants upon COC expansion and gene expression.