Limits...
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.

Show MeSH
Characterization of clonal cell lines. Cell pools isolated by FACS were cloned by limited dilution and GFP expression patterns analyzed by flow cytometry and fluorescence microscopy. (A) Comparison of GFP expression levels (mean fluorescence) of randomly chosen clones, derived either from G418 selection or FACS as outlined in Figure 1. The background autofluorescence of untransfected cells is indicated by a dashed line. (B) Analysis of transgene expression patterns (homogenously positive/heterogeneously positive/negative) according to microscopic analysis of GFP signals. The left panel shows the results for a linearized pCMV-EGFP vector, the middle panel for a corresponding expression cassette devoid of all plasmid backbone sequences. The right panel shows the results for such an expression cassette incorporating the EF promoter instead of the CMV promoter. The sample numbers of individual clones analyzed is indicated. (C) Clones derived from transfections with either EF or CMV expression units as indicated and that were judged homogenously positive by microscopy were analyzed for homogeneity of transgene expression over time. The different time points of flow cytometric GFP profiling during continuous culturing of three independent clones for each experiment are indicated. The upper two panels depict HeLa cell clones, the lower panel CHO cell clones, with the one labeled by an asterisk used for quantitative immunoblotting.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553579&req=5

Figure 2: Characterization of clonal cell lines. Cell pools isolated by FACS were cloned by limited dilution and GFP expression patterns analyzed by flow cytometry and fluorescence microscopy. (A) Comparison of GFP expression levels (mean fluorescence) of randomly chosen clones, derived either from G418 selection or FACS as outlined in Figure 1. The background autofluorescence of untransfected cells is indicated by a dashed line. (B) Analysis of transgene expression patterns (homogenously positive/heterogeneously positive/negative) according to microscopic analysis of GFP signals. The left panel shows the results for a linearized pCMV-EGFP vector, the middle panel for a corresponding expression cassette devoid of all plasmid backbone sequences. The right panel shows the results for such an expression cassette incorporating the EF promoter instead of the CMV promoter. The sample numbers of individual clones analyzed is indicated. (C) Clones derived from transfections with either EF or CMV expression units as indicated and that were judged homogenously positive by microscopy were analyzed for homogeneity of transgene expression over time. The different time points of flow cytometric GFP profiling during continuous culturing of three independent clones for each experiment are indicated. The upper two panels depict HeLa cell clones, the lower panel CHO cell clones, with the one labeled by an asterisk used for quantitative immunoblotting.

Mentions: Next we analyzed clonal cell lines, derived from cell sorted, GFP-positive pools by limited dilution. Their mean fluorescence values were compared with randomly chosen clones obtained by standard antibiotic selection. The results were in line with the analysis of the cell pools, with G418-selected clones being either completely GFP negative, or showing a weak GFP signal, often originating from a limited number of positive cells. In contrast, subcloned isolates from the FACS protocol were all positive for GFP expression according to quantitative flow cytometry (Figure 2A), and the majority of these clones was also unambiguously positive by fluorescence microscopy. We thus analyzed clones derived by the FACS protocol from cells transfected with a linearized pCMV-EGFP vector for their homogeneity according to microscopic inspection. More than one-third of the clones isolated were judged to be uniformly GFP positive (Figure 2B, left panel). In contrast, using the antibiotic selection protocols, we were never able to isolate uniformly expressing clones from stably transfected HeLa cells [see also (21) for comparison]. The ability to recover nonvariegated cell clones with high confidence allowed us to address various parameters frequently discussed to have a major impact on the formation of mosaic expression patterns. First, we asked if bacterial vector sequences have a major influence on transgene expression characteristics. To this end we compared the results obtained for the linearized pCMV-EGFP vector with that from a CMV-EGFP expression cassette. The isolated CMV-EGFP transcription unit (i.e. promoter, reporter gene and polyadenylation signal) devoid of bacterial vector sequences was transfected and cell lines were derived after FACS and subcloning by limited dilution. Figure 2B, middle panel, shows that a higher percentage of homogenous GFP expressing clones could be recovered. Second, the efficiency of obtaining such clones was further improved by using a corresponding EF-EGFP cassette, where the viral CMV promoter was exchanged for the cellular EF1α promoter. This yielded about 80% uniformly transgene-positive clones (Figure 2B, right panel). Some of these latter clones were tested for the uniform expression persistence. They proved to be remarkably stable over >100 PDs (Figure 2C, upper panel), similar to clones obtained after transfection of the CMV-EGFP cassette (Figure 2C, middle panel). Note that these cells do not contain a selectable resistance marker. Thus, both CMV and EF1α promoters can result in production of homogenous, long-term expressing clones, although the EF1α promoter has a higher efficiency. Interestingly, clones expressing the GFP gene under EF1α control showed a higher uniformity in the fluorescence signal as compared to CMV-driven GFP-positive cell clones. The same results for stability and homogeneity of transgene expression were found when the experiments were repeated with CHO cells using the linearized pCMV-EGFP vector (Figure 2C, lower panel). To get a better idea about the absolute expression levels observed, we determined in one of the three CHO clones analyzed the EGFP levels by quantitative immunoblot analysis. In this clone (indicated in Figure 2C, lower panel) the level of EGFP was about 0.5 pg per cell (about 107 molecules/cell).Figure 2.


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)

Characterization of clonal cell lines. Cell pools isolated by FACS were cloned by limited dilution and GFP expression patterns analyzed by flow cytometry and fluorescence microscopy. (A) Comparison of GFP expression levels (mean fluorescence) of randomly chosen clones, derived either from G418 selection or FACS as outlined in Figure 1. The background autofluorescence of untransfected cells is indicated by a dashed line. (B) Analysis of transgene expression patterns (homogenously positive/heterogeneously positive/negative) according to microscopic analysis of GFP signals. The left panel shows the results for a linearized pCMV-EGFP vector, the middle panel for a corresponding expression cassette devoid of all plasmid backbone sequences. The right panel shows the results for such an expression cassette incorporating the EF promoter instead of the CMV promoter. The sample numbers of individual clones analyzed is indicated. (C) Clones derived from transfections with either EF or CMV expression units as indicated and that were judged homogenously positive by microscopy were analyzed for homogeneity of transgene expression over time. The different time points of flow cytometric GFP profiling during continuous culturing of three independent clones for each experiment are indicated. The upper two panels depict HeLa cell clones, the lower panel CHO cell clones, with the one labeled by an asterisk used for quantitative immunoblotting.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Characterization of clonal cell lines. Cell pools isolated by FACS were cloned by limited dilution and GFP expression patterns analyzed by flow cytometry and fluorescence microscopy. (A) Comparison of GFP expression levels (mean fluorescence) of randomly chosen clones, derived either from G418 selection or FACS as outlined in Figure 1. The background autofluorescence of untransfected cells is indicated by a dashed line. (B) Analysis of transgene expression patterns (homogenously positive/heterogeneously positive/negative) according to microscopic analysis of GFP signals. The left panel shows the results for a linearized pCMV-EGFP vector, the middle panel for a corresponding expression cassette devoid of all plasmid backbone sequences. The right panel shows the results for such an expression cassette incorporating the EF promoter instead of the CMV promoter. The sample numbers of individual clones analyzed is indicated. (C) Clones derived from transfections with either EF or CMV expression units as indicated and that were judged homogenously positive by microscopy were analyzed for homogeneity of transgene expression over time. The different time points of flow cytometric GFP profiling during continuous culturing of three independent clones for each experiment are indicated. The upper two panels depict HeLa cell clones, the lower panel CHO cell clones, with the one labeled by an asterisk used for quantitative immunoblotting.
Mentions: Next we analyzed clonal cell lines, derived from cell sorted, GFP-positive pools by limited dilution. Their mean fluorescence values were compared with randomly chosen clones obtained by standard antibiotic selection. The results were in line with the analysis of the cell pools, with G418-selected clones being either completely GFP negative, or showing a weak GFP signal, often originating from a limited number of positive cells. In contrast, subcloned isolates from the FACS protocol were all positive for GFP expression according to quantitative flow cytometry (Figure 2A), and the majority of these clones was also unambiguously positive by fluorescence microscopy. We thus analyzed clones derived by the FACS protocol from cells transfected with a linearized pCMV-EGFP vector for their homogeneity according to microscopic inspection. More than one-third of the clones isolated were judged to be uniformly GFP positive (Figure 2B, left panel). In contrast, using the antibiotic selection protocols, we were never able to isolate uniformly expressing clones from stably transfected HeLa cells [see also (21) for comparison]. The ability to recover nonvariegated cell clones with high confidence allowed us to address various parameters frequently discussed to have a major impact on the formation of mosaic expression patterns. First, we asked if bacterial vector sequences have a major influence on transgene expression characteristics. To this end we compared the results obtained for the linearized pCMV-EGFP vector with that from a CMV-EGFP expression cassette. The isolated CMV-EGFP transcription unit (i.e. promoter, reporter gene and polyadenylation signal) devoid of bacterial vector sequences was transfected and cell lines were derived after FACS and subcloning by limited dilution. Figure 2B, middle panel, shows that a higher percentage of homogenous GFP expressing clones could be recovered. Second, the efficiency of obtaining such clones was further improved by using a corresponding EF-EGFP cassette, where the viral CMV promoter was exchanged for the cellular EF1α promoter. This yielded about 80% uniformly transgene-positive clones (Figure 2B, right panel). Some of these latter clones were tested for the uniform expression persistence. They proved to be remarkably stable over >100 PDs (Figure 2C, upper panel), similar to clones obtained after transfection of the CMV-EGFP cassette (Figure 2C, middle panel). Note that these cells do not contain a selectable resistance marker. Thus, both CMV and EF1α promoters can result in production of homogenous, long-term expressing clones, although the EF1α promoter has a higher efficiency. Interestingly, clones expressing the GFP gene under EF1α control showed a higher uniformity in the fluorescence signal as compared to CMV-driven GFP-positive cell clones. The same results for stability and homogeneity of transgene expression were found when the experiments were repeated with CHO cells using the linearized pCMV-EGFP vector (Figure 2C, lower panel). To get a better idea about the absolute expression levels observed, we determined in one of the three CHO clones analyzed the EGFP levels by quantitative immunoblot analysis. In this clone (indicated in Figure 2C, lower panel) the level of EGFP was about 0.5 pg per cell (about 107 molecules/cell).Figure 2.

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