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M.R. Rodway1 *, C.E. Card1 *, G.B. Sherman2 *, P.J. Chedrese1 *
Department of Obstetrics, Gynecology and Reproductive Sciences, and Department of Herd Medicine and Theriogenology, WCVM, University of Saskatchewan, CANADA 1
Great Plains Veterinary Education Center, University of Nebraska, USA 2
FSH is a 34-kDa glycoprotien hormone consisting of a non–covalently linked heterodimer of two polypeptide chains, called the - and -subunits. This hormone, produced and secreted by the anterior pituitary, is essential for gametogenesis, follicular development and ovulation. The specific objective of this study was to produce recombinant equine FSH. The complete coding region of the equine gonadotropin -subunit gene was amplified by PCR with primers in exon 2 just upstream from the translational start codon and in exon 4 in the 3' untranslated region. The equine FSH -subunit cDNA was isolated by RT–PCR from equine pituitary RNA. The RT primer was a gene-specific primer located 3 kb upstream from the poly A+ tail. PCR primers spanned the entire coding region of the equine FSH -subunit cDNA. Five PCR products from separate RT and PCR reactions were obtained and sequenced to confirm the native sequence of equine FSH -subunit cDNA. The gonadotropin -subunit gene and the FSH -subunit cDNA were each subcloned into pDRSV5.1, a mammalian expression vector containing the Rous sarcoma virus promoter and the dihydrofolate reductase (DHFR) gene. Plasmids were co–transfected into DHFR deficient Chinese hamster ovary (CHO) cells with calcium phosphate or Lipofectin®. A large number of CHO cell lines express the equine gonadotropin - and/or FSH -subunit transgenes, as determined by Northern blot analysis. Recombinant equine FSH glycoprotein produced by these cells is being characterized. Supported by a grant to PJC from the Saskatchewan Agricultural Development Fund.
This abstract is being presented on Sunday, August 1 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.