title: Transient ectopic expression as a method to detect genes conferring drug resistance.
abstract: Ectopic expression provides an alternative to RNAi to conduct functional genomic screens. We have determined whether transient expression can be used to identify genes which confer drug resistance. We constructed a bigenic vector that allows high throughput cloning and which also encodes a red fluorescent protein to monitor transfection. Assay conditions were optimized to allow detection of changes in sensitivity to carboplatin or paclitaxel. Following transient expression of MDR-1 and MCJ, changes in the sensitivity of Sk-Ov-3 cells to paclitaxel were detected whereas expression of Src, Bcl-2 and Bcl-X(L) decreased the sensitivity of Sk-Ov-3 cells to carboplatin. This approach may potentially be used as an independent screen or as a method to help rank hit identified in screens utilizing methods such as RNAi.
authors.affiliation: Section of Medicine, The Institute of Cancer Research, Sutton, United Kingdom.
authors.forename: James
authors.initials: J
authors.lastname: Witham
authors.forename: Susanne
authors.initials: S
authors.lastname: Vidot
authors.forename: Roshan
authors.initials: R
authors.lastname: Agarwal
authors.forename: Stanley B
authors.initials: SB
authors.lastname: Kaye
authors.forename: Alan
authors.initials: A
authors.lastname: Richardson
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D016190:Carboplatin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D019450:Genes, MDR; D019254:Genes, bcl-2; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000235:genetics*; D017239:Paclitaxel / Q000494:pharmacology; D014162:Transfection; D051020:bcl-X Protein / Q000235:genetics; D019061:src-Family Kinases / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016190:Carboplatin; D005456:Fluorescent Dyes; D050956:HSP40 Heat-Shock Proteins; D008164:Luminescent Proteins; D017239:Paclitaxel; D051020:bcl-X Protein; D019061:src-Family Kinases; D000097573:Red Fluorescent Protein; C513055:ABCB1 protein, human; C519405:DNAJC15 protein, human