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PubMed · 10675627

Combinatorial gene expression using multiple episomal vectors.

R A Horlick·A E Schilling·P Samama·R N Swanson·V D Fitzpatrick·A K Robbins·B Damaj
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Abstract

Episomal vectors offer a powerful alternative to integrative recombination for transgene expression in mammalian cells. In this study, various combinations of G protein-coupled receptors (GPCRs) and the G protein subunit G(i2)alpha, were stably expressed from separate episomal vectors in 293-EBNA (293E) cells. Each episome did not adversely affect the others, as gauged by episomal copy number, steady-state mRNA levels and the presence of functional receptors and G protein. Cell lines expressing genes from multiple autonomously replicating vectors were stable just two weeks after transfection, and remained stable in continuous culture for at least 5months. Co-expression of supplementary G(i2)alpha with receptor amplifies the magnitude of signal transduction thereby permitting the development of more sensitive high throughput functional assays. Given these results, combinatorial transfection is the strategy of choice for generating stable cell lines expressing multiple genes for the study of signal-transduction pathways or the evaluation of receptor ligands.

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Blotting, NorthernBlotting, SouthernCalciumCell Line, TransformedChemokine CCL22Chemokine CXCL12Chemokines, CCChemokines, CXCDNAGTP-Binding Protein alpha Subunit, Gi2GTP-Binding Protein alpha Subunits, Gi-GoGene DosageGene Expression RegulationGenetic VectorsHeterotrimeric GTP-Binding ProteinsHumansIodine RadioisotopesPlasmidsProto-Oncogene ProteinsRNARadioligand AssayReceptors, CCR4Receptors, CXCR4Receptors, ChemokineReceptors, OpioidRecombinant Fusion ProteinsTransfectionNociceptin ReceptorJournal Article

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R A Horlick, A E Schilling, P Samama, R N Swanson, V D Fitzpatrick, A K Robbins, B Damaj. 2000-02-08. Combinatorial gene expression using multiple episomal vectors.. https://doi.org/10.1016/s0378-1119(99)00561-2

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