Transient ectopic expression as a method to detect genes conferring drug resistance.
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.
Witham
James
J
Section of Medicine, The Institute of Cancer Research, Sutton, United Kingdom.
Vidot
Susanne
S
Agarwal
Roshan
R
Kaye
Stanley B
SB
Richardson
Alan
A
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
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