title: ER-alpha-cDNA as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones.
abstract: Within the large group of Estrogen Receptor alpha (ERα)-negative breast cancer patients, there is a subgroup carrying the phenotype ERα(-), PR(-), and Her2(-), named accordingly "Triple-Negative" (TN). Using cell lines derived from this TN group, we wished to establish cell clones, in which ERα is ectopically expressed, forming part of a synthetic lethality screening system. Initially, we generated cell transfectants expressing a mono-cistronic ERα transcription unit, adjacent to a separate dominant selectable marker transcription unit. However, the yield of ERα expressing colonies was rather low (5-12.5%), and only about half of these displayed stable ectopic ERα expression over time. Generation and maintenance of such cell clones under minimal exposure to the ERα ligand, did not improve yield or expression stability. Indeed, other groups have also reported grave difficulties in obtaining ectopic expression of ERα in ERα-deficient breast carcinoma cells. We therefore switched to transfecting these cell lines with pERα-IRES, a plasmid vector encoding a bicistronic translation mRNA template: ERα Open Reading Frame (ORF) being upstream followed by a dominant-positive selectable marker (hygro(R)) ORF, directed for translation from an Internal Ribosome Entry Site (IRES). Through usage of this bicistronic vector linkage system, it was possible to generate a very high yield of ERα expressing cell clones (50-100%). The stability over time of these clones was also somewhat improved, though variations between individual cell clones were evident. Our successful experience with ERα in this system may serve as a paradigm for other genes where ectopic expression meets similar hardships.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Shenfeld
authors.forename: Yafit
authors.initials: Y
authors.lastname: Hachmo
authors.forename: Moran
authors.initials: M
authors.lastname: Frenkel
authors.forename: Naomi
authors.initials: N
authors.lastname: Dafni
authors.forename: Michael
authors.initials: M
authors.lastname: Boettcher
authors.forename: Joerg D
authors.initials: JD
authors.lastname: Hoheisel
authors.forename: Iris
authors.initials: I
authors.lastname: Dotan
authors.forename: Dan
authors.initials: D
authors.lastname: Canaani
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D002999:Clone Cells / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D047628:Estrogen Receptor alpha / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D018076:DNA, Complementary; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D008024:Ligands; D012333:RNA, Messenger; D008156:Luciferases