title: Expression efficiency of the human thrombomodulin-encoding gene in various vector and host systems.
abstract: Expression systems were developed for evaluating recombinant human thrombomodulin (TM) production in different host cell lines by investigating the performance of five mammalian expression vectors. The expression vectors were constructed so that they contain multiple monocistronic gene cassettes which include a gene encoding a dominant selectable marker, HyR (hygromycin B phosphotransferase), under the regulation of the thymidine kinase promoter, the target gene which encodes a truncated human re-TM under the regulation of various promoters, an amplifiable gene (Dhfr) encoding murine dihydrofolate reductase under the regulation of either the SV40 early or late promoter along with the SV40 enhancer and the SV40 ori. We tested the performance of the five expression vectors in human embryonic kidney cells (HEK293), baby hamster kidney cells (BHK), human melanoma cells (CHL-1) and Dhfr- Chinese hamster ovary cells (CHO/Dhfr-). We found that the efficiency of DNA uptake, transient expression and stable expression of the different expression vectors were all cell-line dependent. However, the myeloproliferative sarcoma virus (MPSV) LTR promoter consistently showed higher expression levels in all cell lines, particularly in HEK293 cells. These results were confirmed by the distribution curves of the level of expression of individual clones. Furthermore, by amplifying Dhfr in transfected CHO/Dhfr- cells with 100 nM methotrexate, we achieved a 20-fold increase in re-TM production using the SV40 late promoter to control murine Dhfr expression. Our data from DNA and mRNA analysis reveal that pMPSV-TM has a high transcription efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Cardiovascular Research, Berlex Biosciences, Richmond, CA 94804.
authors.forename: J H
authors.initials: JH
authors.lastname: Lin
authors.forename: M
authors.initials: M
authors.lastname: Wang
authors.forename: W H
authors.initials: WH
authors.lastname: Andrews
authors.forename: R
authors.initials: R
authors.lastname: Wydro
authors.forename: J
authors.initials: J
authors.lastname: Morser
mesh_terms: D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA; D005784:Gene Amplification; D005822:Genetic Vectors; D006801:Humans; D011401:Promoter Regions, Genetic; D012313:RNA; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D018180:Thrombomodulin / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D018180:Thrombomodulin; D012313:RNA; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase