|Keller roberts, Madeline|
Submitted to: Biology of Reproduction Abstracts
Publication Type: Abstract only
Publication Acceptance Date: 4/23/1999
Publication Date: N/A
Citation: Keller Roberts, M., Lund, L.A., Ford, J.J., Stocco, D.M., Sherman, G.B. 1999. Expression of recombinant white rhinoceros LH using a dual expression vector [abstract]. Biology of Reproduction Abstracts. 60 (Supplement 1):117. Interpretive Summary:
Technical Abstract: We now report cloning of a 700 bp cDNA encoding white rhinoceros (wr) glycoprotein hormone alpha subunit (wralpha). Deduced positions of 10 highly conserved half-cystine residues and 2 N-glycosylation sites are in agreement with those reported for other species' alpha subunits. Similarity comparisons with a sequences of other vertebrate species [porcine (92.5%), rat (85.7%), and frog (69.9%)] are consistent with expected phylogenetic relationships. Double expression vector, pDEValphabeta, was constructed to co-express wralpha and wrbeta, and includes the dihydrofolate reductase (DHFR) minigene for selection of stable clones. Following confirmational sequencing, pDEValphabeta was transfected into DHFR-deficient Chinese hamster ovary cells (CHO), and conditioned media were collected from confluent cultures of stable clones. Immunoreactive LH-beta 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 immuno-positive for LH-beta. Using this LH-specific bioassay, 4 clones stimulated P secretion (1.6 to 4.1-fold above baseline). All 4 cell lines will be subjected to methotrexate-induced transgene amplification to enhance rec-wrLH expression efficiency.