• Authors: Shenfeld, M., Hachmo, Y., Frenkel, M., Dafni, N., Boettcher, M., Hoheisel, J. D., Dotan, I., Canaani, D.
  • Year: 2012
  • Journal: PLoS One 7 e31977
  • Applications: in vitro / DNA / jetPEI
  • Cell types:
    1. Name: GILM2
      Description: Human breast carcinoma cells.
    2. Name: MDA-MB-231
      Description: Human breast adenocarcinoma cells
      Known as: MDAMB231
    3. Name: MDA-MB-435
      Description: Human breast cancer cells


Within the large group of Estrogen Receptor alpha (ERalpha)-negative breast cancer patients, there is a subgroup carrying the phenotype ERalpha(-), PR(-), and Her2(-), named accordingly "Triple-Negative" (TN). Using cell lines derived from this TN group, we wished to establish cell clones, in which ERalpha is ectopically expressed, forming part of a synthetic lethality screening system. Initially, we generated cell transfectants expressing a mono-cistronic ERalpha transcription unit, adjacent to a separate dominant selectable marker transcription unit. However, the yield of ERalpha expressing colonies was rather low (5-12.5%), and only about half of these displayed stable ectopic ERalpha expression over time. Generation and maintenance of such cell clones under minimal exposure to the ERalpha ligand, did not improve yield or expression stability. Indeed, other groups have also reported grave difficulties in obtaining ectopic expression of ERalpha in ERalpha-deficient breast carcinoma cells. We therefore switched to transfecting these cell lines with pERalpha-IRES, a plasmid vector encoding a bicistronic translation mRNA template: ERalpha 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 ERalpha 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 ERalpha in this system may serve as a paradigm for other genes where ectopic expression meets similar hardships.