title: Analysis of co-transfected expression vectors amplified by methotrexate selection in rat-1 cells.
abstract: In an earlier investigation of the influence of high level expression of p21H-ras, rat-1 cells were co-transfected with a selectable vector (pSV2Neo), an amplifiable vector (encoding dihydrofolate reductase; DHFR) and an H-ras expression vector. In this study we have analyzed the gene dose and expression levels of the three co-transfected plasmid vectors in cell lines that had been selected and isolated at different methotrexate concentrations. Growth of the cells in the absence of selection and Southern blot analyses indicate that the transfected vectors are stably co-integrated into the host genome. High expression levels from all three co-transfected vectors were evident at both the mRNA and protein levels, indicating that they are tightly linked in the host genome. The presence of a large amount of unspliced H-ras mRNA in cells expressing high levels of H-ras p21 indicates that processing of mRNA may be rate-limiting. Comparison of the gene dose and expression levels shows that the resistance of cells to increased methotrexate concentrations can occur by different mechanisms. It is concluded that co-transfection of individual plasmid vectors into rat-1 cells, followed by methotrexate selection, is an effective manner of achieving high level expression of proteins in cultured cells.
authors.affiliation: University of Stellenbosch (US)/Medical Research Council (MRC) Center for Molecular and Cellular Biology, Tygerberg, South Africa.
authors.forename: M H
authors.initials: MH
authors.lastname: Ricketts
authors.forename: S
authors.initials: S
authors.lastname: Vandenplas
authors.forename: M J
authors.initials: MJ
authors.lastname: van der Merwe
authors.forename: D U
authors.initials: DU
authors.lastname: Bellstedt
authors.forename: H M
authors.initials: HM
authors.lastname: North
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics; D005784:Gene Amplification / Q000187:drug effects* / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D005819:Genetic Markers; D005822:Genetic Vectors; D008727:Methotrexate / Q000494:pharmacology*; D009355:Neomycin / Q000494:pharmacology; D014176:Protein Biosynthesis; D016283:Proto-Oncogene Proteins p21(ras) / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection / Q000235:genetics*
chemical_list: D005819:Genetic Markers; D012333:RNA, Messenger; D013762:Tetrahydrofolate Dehydrogenase; D016283:Proto-Oncogene Proteins p21(ras); D009355:Neomycin; D008727:Methotrexate