Abstract: 77
EXPRESSION OF RECOMBINANT WHITE RHINOCEROS LH USING A DUAL EXPRESSION VECTOR.
M Keller Roberts1 , L.A. Lund2 , J.J. Ford3 *, D.M. Stocco4 *, G.B. Sherman1 *
Department of Veterinary and Biomedical Sciences, University of Nebraska, Clay Center, NE 1
Department of Veterinary Biosciences, University of Illinois, Urbana, IL 2
USDA-ARS, US Meat Animal Research Center, Clay Center, NE 3
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 4
Expression and purification of recombinant (rec–) white rhinoceros (wr) LH will be essential for developing sensitive, non-invasive assays to monitor rhino fertility. We previously isolated cDNA and genomic clones encoding wr LH 
subunit (wr) (Lund and Sherman, 1998). We now report cloning of a 700bp cDNA encoding wr glycoprotein hormone 
subunit (wr). Deduced positions of 10 highly conserved half-cystine residues and 2 N-glycosylation sites are in agreement with those reported for other species' subunits. Similarity comparisons with sequences of other vertebrate species [porcine (92.5%), rat (85.7%), and frog (69.9%)] are consistent with expected phylogenetic relationships, providing evidence that the cDNA reported herein encodes bona fide wr.
Double expression vector, pDEV, was constructed to co-express wr and wr, and includes the dihydrofolate reductase (DHFR) minigene for selection of stable clones. Following confirmational sequencing, pDEVa was transfected into DHFR-deficient Chinese hamster ovary cells (CHO), and conditioned media were collected from confluent cultures of stable clones. Immunoreactive LH was detected in media from 11/36 CHO clones by heterologous RIA using monoclonal antibody 518B7. Values ranged from non-detectable to 12.7 ng/ml. Presence of functional rec-wrLH heterodimer was evaluated by progesterone (P) response of MA–10 Leydig tumor cells to media from 8 clones immunopositive for LH. Using this LH-specific bioassay, 4 clones stimulated P secretion (1.6 to 4.1–fold above baseline). All cell lines will be subjected to methotrexate–induced transgene amplification to enhance rec-wrLH expression efficiency. Supported by Morris Animal Foundation, grant 96ZO-25.
This abstract is being presented on Sunday, August 1 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.