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

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 whic...

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Autores principales:	Michal Shenfeld, Yafit Hachmo, Moran Frenkel, Naomi Dafni, Michael Boettcher, Joerg D Hoheisel, Iris Dotan, Dan Canaani
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/71e97a1b778a45fa85e7dae720efc2f9
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spelling	oai:doaj.org-article:71e97a1b778a45fa85e7dae720efc2f92021-11-18T07:27:27ZER-alpha-cDNA as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones.1932-620310.1371/journal.pone.0031977https://doaj.org/article/71e97a1b778a45fa85e7dae720efc2f92012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22363779/?tool=EBIhttps://doaj.org/toc/1932-6203Within 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.Michal ShenfeldYafit HachmoMoran FrenkelNaomi DafniMichael BoettcherJoerg D HoheiselIris DotanDan CanaaniPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 2, p e31977 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Michal Shenfeld Yafit Hachmo Moran Frenkel Naomi Dafni Michael Boettcher Joerg D Hoheisel Iris Dotan Dan Canaani ER-alpha-cDNA as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones.
description	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.
format	article
author	Michal Shenfeld Yafit Hachmo Moran Frenkel Naomi Dafni Michael Boettcher Joerg D Hoheisel Iris Dotan Dan Canaani
author_facet	Michal Shenfeld Yafit Hachmo Moran Frenkel Naomi Dafni Michael Boettcher Joerg D Hoheisel Iris Dotan Dan Canaani
author_sort	Michal Shenfeld
title	ER-alpha-cDNA as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones.
title_short	ER-alpha-cDNA as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones.
title_full	ER-alpha-cDNA as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones.
title_fullStr	ER-alpha-cDNA as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones.
title_full_unstemmed	ER-alpha-cDNA as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones.
title_sort	er-alpha-cdna as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/71e97a1b778a45fa85e7dae720efc2f9
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ER-alpha-cDNA as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones.

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