Combinatorial gene expression using multiple episomal vectors.
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.
Horlick
R A
RA
Department of Drug Discovery, Pharmacopeia, Inc., CN5350, Princeton, NJ, USA. horlick@mpi.com
Schilling
A E
AE
Samama
P
P
Swanson
R N
RN
Fitzpatrick
V D
VD
Robbins
A K
AK
Damaj
B
B
D015152:Blotting, Northern; D015139:Blotting, Southern; D002118:Calcium / Q000378:metabolism; D002461:Cell Line, Transformed; D054422:Chemokine CCL22; D054377:Chemokine CXCL12; D019742:Chemokines, CC / Q000378:metabolism; D019743:Chemokines, CXC / Q000378:metabolism; D004247:DNA / Q000235:genetics; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D018628:Gene Dosage; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D010957:Plasmids / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics; D011869:Radioligand Assay; D054398:Receptors, CCR4; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism; D011957:Receptors, Opioid / Q000819:agonists / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection; D000094942:Nociceptin Receptor
C515482:CCL22 protein, human; C515354:CCR4 protein, human; C515255:CXCL12 protein, human; D054422:Chemokine CCL22; D054377:Chemokine CXCL12; D019742:Chemokines, CC; D019743:Chemokines, CXC; D007457:Iodine Radioisotopes; D011518:Proto-Oncogene Proteins; D054398:Receptors, CCR4; D019718:Receptors, CXCR4; D019707:Receptors, Chemokine; D011957:Receptors, Opioid; D011993:Recombinant Fusion Proteins; D012313:RNA; D004247:DNA; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium; D000094942:Nociceptin Receptor; C000722283:OPRL1 protein, human