Expression of the genes encoding bovine LH in a line of Chinese hamster ovary cells.
Synthesis of biologically active LH is complex, due in part to its heterodimeric subunit structure and to the numerous post-translation modifications of each subunit. Through the use of mammalian expression vectors we have been able to introduce the bovine alpha subunit and LH-beta genes into a Chinese hamster ovary cell line deficient in dihydrofolate reductase. The bovine genes are actively expressed and the Chinese hamster ovary cells secrete biologically active LH. The expression vector containing the bovine alpha subunit gene also contains a modified mouse gene encoding dihydrofolate reductase, permitting the use of methotrexate to amplify selectively the bovine alpha subunit gene after its integration into the genome of the Chinese hamster cells. This provides a novel means for assessing the importance of alpha subunit concentration with respect to assembly of the heterodimer. In addition, methotrexate selection leads to the over-production of LH (10 micrograms/10(6) cells/24 h). Finally, because the bovine LH produced in the Chinese hamster ovary cells is glycosylated, this transfection system can be used in conjunction with in-vitro mutagenesis to determine whether site-specific changes in glycosylation have an effect on subunit assembly and biological activity. This transfection approach therefore offers multiple avenues to explore further the molecular mechanisms underlying the complex biosynthetic pathway of bovine LH.
D000818:Animals; D002417:Cattle / Q000235:genetics*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D007986:Luteinizing Hormone / Q000235:genetics*; D008727:Methotrexate / Q000494:pharmacology; D010053:Ovary / Q000166:cytology
D007986:Luteinizing Hormone; D008727:Methotrexate