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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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Recombinase-mediated switch of transgene expression. (A) Schematic presentation of the expression vectors for FLP-mediated switch of transgene expression used in this study. Genes of interest (GOI) inserted by us in the multiple cloning sites (mcs) were oct4 and mRFP. The deletion principle is outlined for the latter transgene, with the transcripts before and after recombination indicated. (B) Cells stably transfected with pEFF3EGFPF3mRFP were followed over the time course of a deletion experiment, showing both the GFP and RFP signals. Left panel: only EGFP was expressed. Middle panel: in cells where FLP levels upon transient transfection were sufficient to promote EGFP deletion, mRFP expression was activated. Untransfected cells stayed green. Right panel: recloning after FLP expression showed that GFP-negative clones were uniformly positive for RFP. (C) Southern blot analysis of a selected pEFF3EGFPF3mRFP clone before FLP-mediated recombination and a corresponding subclone after deletion of the EGFP reporter. The hybridization probe used is the mRFP gene. Note that the signal intensity for the mRFP target sequence decreased twofold after recombination (phosphorimager signal 246 versus 137 arbitrary units). The recombination principle anticipates a reduction of inserts to single copy transgenes. (D) The switch from GFP to RFP expression as monitored by flow cytometry.
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Figure 3: Recombinase-mediated switch of transgene expression. (A) Schematic presentation of the expression vectors for FLP-mediated switch of transgene expression used in this study. Genes of interest (GOI) inserted by us in the multiple cloning sites (mcs) were oct4 and mRFP. The deletion principle is outlined for the latter transgene, with the transcripts before and after recombination indicated. (B) Cells stably transfected with pEFF3EGFPF3mRFP were followed over the time course of a deletion experiment, showing both the GFP and RFP signals. Left panel: only EGFP was expressed. Middle panel: in cells where FLP levels upon transient transfection were sufficient to promote EGFP deletion, mRFP expression was activated. Untransfected cells stayed green. Right panel: recloning after FLP expression showed that GFP-negative clones were uniformly positive for RFP. (C) Southern blot analysis of a selected pEFF3EGFPF3mRFP clone before FLP-mediated recombination and a corresponding subclone after deletion of the EGFP reporter. The hybridization probe used is the mRFP gene. Note that the signal intensity for the mRFP target sequence decreased twofold after recombination (phosphorimager signal 246 versus 137 arbitrary units). The recombination principle anticipates a reduction of inserts to single copy transgenes. (D) The switch from GFP to RFP expression as monitored by flow cytometry.

Mentions: We next asked if it is possible to adapt the approach outlined here for the generation of stable cell lines for transgenes that do not convey a sortable cellular phenotype. To this end, we constructed expression units as outlined in Figure 3A. Here, EF1α drives expression of a GFP gene flanked by FRT sites arranged in the same orientation. Upon FLP recombination, the promoter proximal GFP gene and its polyadenylation signal will be deleted and a distal, previously not transcribed gene of interest would be placed under control of the EF promoter. To test this principle in an experimental setting that would allow us to directly monitor the succession of events, we initially chose a red fluorescent protein (RFP) gene. Cells were transfected and sorted for GFP expression and individual clones characterized for homogeneity and persistence of the GFP signal. These RFP-negative cells were transfected with a vector encoding enhanced FLP (19). Recombinase expression caused deletion of GFP and placement of RFP under EF1α control, as indicated by previously green cells turning to red (Figure 3B). The deletion event was also monitored by Southern blotting (Figure 3C). After recloning, all of the GFP-negative clones analyzed were homogenously RFP-positive, and maintained this expression pattern (Figure 3D).Figure 3.


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)

Recombinase-mediated switch of transgene expression. (A) Schematic presentation of the expression vectors for FLP-mediated switch of transgene expression used in this study. Genes of interest (GOI) inserted by us in the multiple cloning sites (mcs) were oct4 and mRFP. The deletion principle is outlined for the latter transgene, with the transcripts before and after recombination indicated. (B) Cells stably transfected with pEFF3EGFPF3mRFP were followed over the time course of a deletion experiment, showing both the GFP and RFP signals. Left panel: only EGFP was expressed. Middle panel: in cells where FLP levels upon transient transfection were sufficient to promote EGFP deletion, mRFP expression was activated. Untransfected cells stayed green. Right panel: recloning after FLP expression showed that GFP-negative clones were uniformly positive for RFP. (C) Southern blot analysis of a selected pEFF3EGFPF3mRFP clone before FLP-mediated recombination and a corresponding subclone after deletion of the EGFP reporter. The hybridization probe used is the mRFP gene. Note that the signal intensity for the mRFP target sequence decreased twofold after recombination (phosphorimager signal 246 versus 137 arbitrary units). The recombination principle anticipates a reduction of inserts to single copy transgenes. (D) The switch from GFP to RFP expression as monitored by flow cytometry.
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Related In: Results  -  Collection

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Figure 3: Recombinase-mediated switch of transgene expression. (A) Schematic presentation of the expression vectors for FLP-mediated switch of transgene expression used in this study. Genes of interest (GOI) inserted by us in the multiple cloning sites (mcs) were oct4 and mRFP. The deletion principle is outlined for the latter transgene, with the transcripts before and after recombination indicated. (B) Cells stably transfected with pEFF3EGFPF3mRFP were followed over the time course of a deletion experiment, showing both the GFP and RFP signals. Left panel: only EGFP was expressed. Middle panel: in cells where FLP levels upon transient transfection were sufficient to promote EGFP deletion, mRFP expression was activated. Untransfected cells stayed green. Right panel: recloning after FLP expression showed that GFP-negative clones were uniformly positive for RFP. (C) Southern blot analysis of a selected pEFF3EGFPF3mRFP clone before FLP-mediated recombination and a corresponding subclone after deletion of the EGFP reporter. The hybridization probe used is the mRFP gene. Note that the signal intensity for the mRFP target sequence decreased twofold after recombination (phosphorimager signal 246 versus 137 arbitrary units). The recombination principle anticipates a reduction of inserts to single copy transgenes. (D) The switch from GFP to RFP expression as monitored by flow cytometry.
Mentions: We next asked if it is possible to adapt the approach outlined here for the generation of stable cell lines for transgenes that do not convey a sortable cellular phenotype. To this end, we constructed expression units as outlined in Figure 3A. Here, EF1α drives expression of a GFP gene flanked by FRT sites arranged in the same orientation. Upon FLP recombination, the promoter proximal GFP gene and its polyadenylation signal will be deleted and a distal, previously not transcribed gene of interest would be placed under control of the EF promoter. To test this principle in an experimental setting that would allow us to directly monitor the succession of events, we initially chose a red fluorescent protein (RFP) gene. Cells were transfected and sorted for GFP expression and individual clones characterized for homogeneity and persistence of the GFP signal. These RFP-negative cells were transfected with a vector encoding enhanced FLP (19). Recombinase expression caused deletion of GFP and placement of RFP under EF1α control, as indicated by previously green cells turning to red (Figure 3B). The deletion event was also monitored by Southern blotting (Figure 3C). After recloning, all of the GFP-negative clones analyzed were homogenously RFP-positive, and maintained this expression pattern (Figure 3D).Figure 3.

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