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
Homogenous expression of Oct4. (A) A homogenously GFP-positive pEFF3EGFPF3hOct4 clone is shown in the top panel. Three days after transfection of a FLP expression vector a bimodal GFP pattern can be recognized (bottom panel), as FLP expressing cells show a progressive loss of the GFP signal upon reporter gene deletion. (B) Immunoblot analysis for Oct4 expression. Neither the parental cell line nor the nonrecombined pEFF3EGFPF3hOct4 clone expressed detectable levels of Oct4. Of the three subclones isolated after FLP-mediated EGFP deletion, two showed a strong Oct4 signal. (C) RNA isolated from the parental cell line, the nonrecombined and a recombined pEFF3EGFPF3hOct4 clone was analyzed for Oct4-specific transcript levels by qPCR. (D) After FLP-mediated recombination, Oct4 (shown in blue) can be found in every nucleus of a pEFF3EGFPF3hOct4 subclone (lower panel), contrary to the initial, nonrecombined pEFF3EGFPF3hOct4 clone (upper panel) that was Oct4 negative. The counter stain in this immunofluorescence analysis was tubulin, shown in yellow.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Homogenous expression of Oct4. (A) A homogenously GFP-positive pEFF3EGFPF3hOct4 clone is shown in the top panel. Three days after transfection of a FLP expression vector a bimodal GFP pattern can be recognized (bottom panel), as FLP expressing cells show a progressive loss of the GFP signal upon reporter gene deletion. (B) Immunoblot analysis for Oct4 expression. Neither the parental cell line nor the nonrecombined pEFF3EGFPF3hOct4 clone expressed detectable levels of Oct4. Of the three subclones isolated after FLP-mediated EGFP deletion, two showed a strong Oct4 signal. (C) RNA isolated from the parental cell line, the nonrecombined and a recombined pEFF3EGFPF3hOct4 clone was analyzed for Oct4-specific transcript levels by qPCR. (D) After FLP-mediated recombination, Oct4 (shown in blue) can be found in every nucleus of a pEFF3EGFPF3hOct4 subclone (lower panel), contrary to the initial, nonrecombined pEFF3EGFPF3hOct4 clone (upper panel) that was Oct4 negative. The counter stain in this immunofluorescence analysis was tubulin, shown in yellow.

Mentions: Having shown the proof of principle, we turned to the expression of human oct4, the homogenous expression of which is of immediate relevance for the ongoing work in our laboratory. The human oct 4 gene was placed behind the FRT-flanked GFP cassette. Generation of stable, homogenous GFP-positive HeLa cell clones with a nontranscribed oct4 gene was as described before. HeLa cells were chosen as they are otherwise negative for oct4 expression. FLP-mediated deletion of the reporter cassette caused a decrease in the GFP signal of the transfected subpopulation (Figure 4A). Subsequent recloning resulted in transgene expression in 2 out of 3 subclones analyzed, as shown by immunoblot analysis for Oct4 (Figure 4B). We corroborated these results by quantitative analysis of the oct4 transcript levels. cDNA obtained from total RNA was analyzed by qPCR. As shown in Figure 4C, the oct4 RNA levels of HeLa cells stably transfected with pEFF3EGFPF3Oct4 was barely above the low levels found in the parental HeLa cells. FLP-mediated cassette deletion resulted in a strong signal for the oct4 transcript. Lastly, the homogeneity of transgene expression was monitored by immunofluorescence analysis for Oct4 protein. Prior to deletion of the GFP cassette, HeLa cells stably transfected with pEFF3EGFPF3Oct4 did not show a discernable Oct4 signal, in line with the immunoblot analysis. In contrast, after FLP-mediated deletion of the reporter cassette, clonal cells were uniformly positive for Oct4, with the transcription factor accumulating in the nucleus (Figure 4D).Figure 4.


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)

Homogenous expression of Oct4. (A) A homogenously GFP-positive pEFF3EGFPF3hOct4 clone is shown in the top panel. Three days after transfection of a FLP expression vector a bimodal GFP pattern can be recognized (bottom panel), as FLP expressing cells show a progressive loss of the GFP signal upon reporter gene deletion. (B) Immunoblot analysis for Oct4 expression. Neither the parental cell line nor the nonrecombined pEFF3EGFPF3hOct4 clone expressed detectable levels of Oct4. Of the three subclones isolated after FLP-mediated EGFP deletion, two showed a strong Oct4 signal. (C) RNA isolated from the parental cell line, the nonrecombined and a recombined pEFF3EGFPF3hOct4 clone was analyzed for Oct4-specific transcript levels by qPCR. (D) After FLP-mediated recombination, Oct4 (shown in blue) can be found in every nucleus of a pEFF3EGFPF3hOct4 subclone (lower panel), contrary to the initial, nonrecombined pEFF3EGFPF3hOct4 clone (upper panel) that was Oct4 negative. The counter stain in this immunofluorescence analysis was tubulin, shown in yellow.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Homogenous expression of Oct4. (A) A homogenously GFP-positive pEFF3EGFPF3hOct4 clone is shown in the top panel. Three days after transfection of a FLP expression vector a bimodal GFP pattern can be recognized (bottom panel), as FLP expressing cells show a progressive loss of the GFP signal upon reporter gene deletion. (B) Immunoblot analysis for Oct4 expression. Neither the parental cell line nor the nonrecombined pEFF3EGFPF3hOct4 clone expressed detectable levels of Oct4. Of the three subclones isolated after FLP-mediated EGFP deletion, two showed a strong Oct4 signal. (C) RNA isolated from the parental cell line, the nonrecombined and a recombined pEFF3EGFPF3hOct4 clone was analyzed for Oct4-specific transcript levels by qPCR. (D) After FLP-mediated recombination, Oct4 (shown in blue) can be found in every nucleus of a pEFF3EGFPF3hOct4 subclone (lower panel), contrary to the initial, nonrecombined pEFF3EGFPF3hOct4 clone (upper panel) that was Oct4 negative. The counter stain in this immunofluorescence analysis was tubulin, shown in yellow.
Mentions: Having shown the proof of principle, we turned to the expression of human oct4, the homogenous expression of which is of immediate relevance for the ongoing work in our laboratory. The human oct 4 gene was placed behind the FRT-flanked GFP cassette. Generation of stable, homogenous GFP-positive HeLa cell clones with a nontranscribed oct4 gene was as described before. HeLa cells were chosen as they are otherwise negative for oct4 expression. FLP-mediated deletion of the reporter cassette caused a decrease in the GFP signal of the transfected subpopulation (Figure 4A). Subsequent recloning resulted in transgene expression in 2 out of 3 subclones analyzed, as shown by immunoblot analysis for Oct4 (Figure 4B). We corroborated these results by quantitative analysis of the oct4 transcript levels. cDNA obtained from total RNA was analyzed by qPCR. As shown in Figure 4C, the oct4 RNA levels of HeLa cells stably transfected with pEFF3EGFPF3Oct4 was barely above the low levels found in the parental HeLa cells. FLP-mediated cassette deletion resulted in a strong signal for the oct4 transcript. Lastly, the homogeneity of transgene expression was monitored by immunofluorescence analysis for Oct4 protein. Prior to deletion of the GFP cassette, HeLa cells stably transfected with pEFF3EGFPF3Oct4 did not show a discernable Oct4 signal, in line with the immunoblot analysis. In contrast, after FLP-mediated deletion of the reporter cassette, clonal cells were uniformly positive for Oct4, with the transcription factor accumulating in the nucleus (Figure 4D).Figure 4.

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