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Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation.

Kaufman WL, Kocman I, Agrawal V, Rahn HP, Besser D, Gossen M - Nucleic Acids Res. (2008)

Bottom Line: They are widely attributed to features of transgenic transcription units distinct from endogenous genes, rendering them particularly susceptible to epigenetic downregulation.Contrary to this assumption we show that the method used for the isolation of stably transfected cells has the most profound impact on transgene expression patterns.However, by combining this approach with site-specific recombination, it can be applied to isolate stable cell lines with the desired expression characteristics for any gene of interest.

View Article: PubMed Central - PubMed

Affiliation: Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.

ABSTRACT
Nonuniform, mosaic expression patterns of transgenes are often linked to transcriptional silencing, triggered by epigenetic modifications of the exogenous DNA. Such phenotypes are common phenomena in genetically engineered cells and organisms. They are widely attributed to features of transgenic transcription units distinct from endogenous genes, rendering them particularly susceptible to epigenetic downregulation. Contrary to this assumption we show that the method used for the isolation of stably transfected cells has the most profound impact on transgene expression patterns. Standard antibiotic selection was directly compared to cell sorting for the establishment of stable cells. Only the latter procedure could warrant a high degree of uniformity and stability in gene expression. Marker genes useful for the essential cell sorting step encode mostly fluorescent proteins. However, by combining this approach with site-specific recombination, it can be applied to isolate stable cell lines with the desired expression characteristics for any gene of interest.

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Flow chart of FACS and antibiotic-selection protocols. HeLa cells were transfected with pEGFP-C1. One day posttransfection (d.p.t.) the cells were divided, with one aliquot subjected to G418 selection to obtain a stably transfected cell population. The other aliquot of cells was grown in the absence of antibiotic selection, and cells were initially subjected to cell sorting for EGFP expressers at 4 d.p.t. The sorting gate and percentage of sorted cells of the whole population is indicated in the right inset showing the FACS profile for the fluorescence signal. Due to the transient nature of EGFP expression at this time point, the majority of the gated population lost the reporter gene expression when grown without antibiotic selection until the second cell sorting at 11 d.p.t. The residual positive population was expanded and analyzed for fluorescent protein expression in parallel with the population grown under drug selection, obtained by pooling all G418-resistant colonies. The FACS profiles at the bottom show the fluorescence signals from the pools of transfected cells (black line) in comparison to untransfected, parental HeLa cells (grey line). Further growth of the sorted population for 30 population doublings without selective pressure resulted only in a minor loss of the EGFP signal (grey-filled area in bottom right FACS profile).
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Figure 1: Flow chart of FACS and antibiotic-selection protocols. HeLa cells were transfected with pEGFP-C1. One day posttransfection (d.p.t.) the cells were divided, with one aliquot subjected to G418 selection to obtain a stably transfected cell population. The other aliquot of cells was grown in the absence of antibiotic selection, and cells were initially subjected to cell sorting for EGFP expressers at 4 d.p.t. The sorting gate and percentage of sorted cells of the whole population is indicated in the right inset showing the FACS profile for the fluorescence signal. Due to the transient nature of EGFP expression at this time point, the majority of the gated population lost the reporter gene expression when grown without antibiotic selection until the second cell sorting at 11 d.p.t. The residual positive population was expanded and analyzed for fluorescent protein expression in parallel with the population grown under drug selection, obtained by pooling all G418-resistant colonies. The FACS profiles at the bottom show the fluorescence signals from the pools of transfected cells (black line) in comparison to untransfected, parental HeLa cells (grey line). Further growth of the sorted population for 30 population doublings without selective pressure resulted only in a minor loss of the EGFP signal (grey-filled area in bottom right FACS profile).

Mentions: To facilitate a direct comparison between antibiotic selection and FACS for the isolation of cells expressing a transgene after chromosomal integration, we first analyzed two cell populations derived from a single transfection experiment. We used a plasmid harboring both a CMV promoter driving expression of the green fluorescent protein (GFP), as well as a transcription unit conferring neomycin resistance. After transfection of the linearized vector into HeLa cells, its dual function enabled antibiotic selection of G418-resistant cells and cell sorting for fluorescent protein expression in parallel experiments. FACS enrichment of GFP-positive cells was done twice and the fluorescence level of the resulting cell population compared directly to the GFP expression of pooled G418-resistant clones originating from the same transfection. Figure 1 shows that contrary to the sorted cell pool, the vast majority of G418-resistant cells showed little to no GFP signal. Microscopic examination of G418-resistant clones prior to pooling had shown that the few GFP-positive cells did not originate from a rare clone with intense GFP expression, but that a low percentage of positive cells was visible in several individual colonies. The high level of GFP expression in the sorted cell pool showed only a minor decrease when reanalyzed after growing those cells for about 30 population doublings (PDs) without applying selective pressure through addition of G418 (Figure 1). All subsequent cell sorting experiments were done according to the basic flow chart outlined in Figure 1. We did not notice any marked difference in the results when making minor adjustments to the parameters indicated.Figure 1.


Homogeneity and persistence of transgene expression by omitting antibiotic selection in cell line isolation.

Kaufman WL, Kocman I, Agrawal V, Rahn HP, Besser D, Gossen M - Nucleic Acids Res. (2008)

Flow chart of FACS and antibiotic-selection protocols. HeLa cells were transfected with pEGFP-C1. One day posttransfection (d.p.t.) the cells were divided, with one aliquot subjected to G418 selection to obtain a stably transfected cell population. The other aliquot of cells was grown in the absence of antibiotic selection, and cells were initially subjected to cell sorting for EGFP expressers at 4 d.p.t. The sorting gate and percentage of sorted cells of the whole population is indicated in the right inset showing the FACS profile for the fluorescence signal. Due to the transient nature of EGFP expression at this time point, the majority of the gated population lost the reporter gene expression when grown without antibiotic selection until the second cell sorting at 11 d.p.t. The residual positive population was expanded and analyzed for fluorescent protein expression in parallel with the population grown under drug selection, obtained by pooling all G418-resistant colonies. The FACS profiles at the bottom show the fluorescence signals from the pools of transfected cells (black line) in comparison to untransfected, parental HeLa cells (grey line). Further growth of the sorted population for 30 population doublings without selective pressure resulted only in a minor loss of the EGFP signal (grey-filled area in bottom right FACS profile).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2553579&req=5

Figure 1: Flow chart of FACS and antibiotic-selection protocols. HeLa cells were transfected with pEGFP-C1. One day posttransfection (d.p.t.) the cells were divided, with one aliquot subjected to G418 selection to obtain a stably transfected cell population. The other aliquot of cells was grown in the absence of antibiotic selection, and cells were initially subjected to cell sorting for EGFP expressers at 4 d.p.t. The sorting gate and percentage of sorted cells of the whole population is indicated in the right inset showing the FACS profile for the fluorescence signal. Due to the transient nature of EGFP expression at this time point, the majority of the gated population lost the reporter gene expression when grown without antibiotic selection until the second cell sorting at 11 d.p.t. The residual positive population was expanded and analyzed for fluorescent protein expression in parallel with the population grown under drug selection, obtained by pooling all G418-resistant colonies. The FACS profiles at the bottom show the fluorescence signals from the pools of transfected cells (black line) in comparison to untransfected, parental HeLa cells (grey line). Further growth of the sorted population for 30 population doublings without selective pressure resulted only in a minor loss of the EGFP signal (grey-filled area in bottom right FACS profile).
Mentions: To facilitate a direct comparison between antibiotic selection and FACS for the isolation of cells expressing a transgene after chromosomal integration, we first analyzed two cell populations derived from a single transfection experiment. We used a plasmid harboring both a CMV promoter driving expression of the green fluorescent protein (GFP), as well as a transcription unit conferring neomycin resistance. After transfection of the linearized vector into HeLa cells, its dual function enabled antibiotic selection of G418-resistant cells and cell sorting for fluorescent protein expression in parallel experiments. FACS enrichment of GFP-positive cells was done twice and the fluorescence level of the resulting cell population compared directly to the GFP expression of pooled G418-resistant clones originating from the same transfection. Figure 1 shows that contrary to the sorted cell pool, the vast majority of G418-resistant cells showed little to no GFP signal. Microscopic examination of G418-resistant clones prior to pooling had shown that the few GFP-positive cells did not originate from a rare clone with intense GFP expression, but that a low percentage of positive cells was visible in several individual colonies. The high level of GFP expression in the sorted cell pool showed only a minor decrease when reanalyzed after growing those cells for about 30 population doublings (PDs) without applying selective pressure through addition of G418 (Figure 1). All subsequent cell sorting experiments were done according to the basic flow chart outlined in Figure 1. We did not notice any marked difference in the results when making minor adjustments to the parameters indicated.Figure 1.

Bottom Line: They are widely attributed to features of transgenic transcription units distinct from endogenous genes, rendering them particularly susceptible to epigenetic downregulation.Contrary to this assumption we show that the method used for the isolation of stably transfected cells has the most profound impact on transgene expression patterns.However, by combining this approach with site-specific recombination, it can be applied to isolate stable cell lines with the desired expression characteristics for any gene of interest.

View Article: PubMed Central - PubMed

Affiliation: Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.

ABSTRACT
Nonuniform, mosaic expression patterns of transgenes are often linked to transcriptional silencing, triggered by epigenetic modifications of the exogenous DNA. Such phenotypes are common phenomena in genetically engineered cells and organisms. They are widely attributed to features of transgenic transcription units distinct from endogenous genes, rendering them particularly susceptible to epigenetic downregulation. Contrary to this assumption we show that the method used for the isolation of stably transfected cells has the most profound impact on transgene expression patterns. Standard antibiotic selection was directly compared to cell sorting for the establishment of stable cells. Only the latter procedure could warrant a high degree of uniformity and stability in gene expression. Marker genes useful for the essential cell sorting step encode mostly fluorescent proteins. However, by combining this approach with site-specific recombination, it can be applied to isolate stable cell lines with the desired expression characteristics for any gene of interest.

Show MeSH
Related in: MedlinePlus