ER-alpha-cDNA as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones.

PloS One
Michal ShenfeldDan Canaani

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...Continue Reading

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Citations

Jun 3, 2021·International Journal of Molecular Sciences·Guus Gijsbertus Hubert van den AkkerTim Johannes Maria Welting

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Methods Mentioned

BETA
transfection
transfections
FCS
Assay
PCR

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