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Characterization of parameters required for effective use of tamoxifen-regulated recombination.

Buelow B, Scharenberg AM - PLoS ONE (2008)

Bottom Line: In the DT40 chicken B cell line, we generated an mCrem-based reversible switch for conditional regulation of a transgene, and in contrast with previous work, observed significant constitutive activity of mCrem.This prompted us to use our system for analysis of the parameters governing tamoxifen-regulated mCrem recombination of a genomic target.We also observe time and dose-dependent effects on mCrem activity which suggest limitations on the use of conditional targeting approaches for applications which require tight temporal coordination of Cre action within a cell population.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Washington, Seattle Children's Hospital Research Institute, Seattle, Washington, USA.

ABSTRACT
Conditional gene targeting using the Cre-loxp system is a well established technique in numerous in vitro and in vivo systems. Ligand regulated forms of Cre have been increasingly used in these applications in order to gain temporal and spatial control over conditional targeting. The tamoxifen-regulated Cre variant mer-Cre-mer (mCrem) is widely utilized because of its reputation for tight regulation in the absence of its tamoxifen ligand. In the DT40 chicken B cell line, we generated an mCrem-based reversible switch for conditional regulation of a transgene, and in contrast with previous work, observed significant constitutive activity of mCrem. This prompted us to use our system for analysis of the parameters governing tamoxifen-regulated mCrem recombination of a genomic target. We find that robust mCrem expression correlates with a high level of tamoxifen-independent Cre activity, while clones expressing mCrem at the limit of western blot detection exhibit extremely tight regulation. We also observe time and dose-dependent effects on mCrem activity which suggest limitations on the use of conditional targeting approaches for applications which require tight temporal coordination of Cre action within a cell population.

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Sustained OHT-independent flipping of the floxed cassette in 27Flip/Cre20 cells correlates with mer-Cre-mer expression level.A Following stable transfection of 27Flip cells with an mCrem expressing vector, 5 clones expressing various amounts of mCrem were selected. Cells from the clones were lysed and 50 µg of protein of each were run on an 8% SDS-PAGE gel. MCrem was visualized with polyclonal rabbit anti-Cre antibody (1∶2000, Novagen), with β-actin visualized with a polyclonal mouse antibody (1∶40000, Sigma) as a loading control. 27Flip/Cre15 and -20 were designated “high expressors”, and 27Flip/Cre26, -31, and -35 were designated “low expressors”. B 27Flip/Cre clones were either left untreated or treated with 1 µM OHT for 48 hours and then allowed to grow in culture for 18 days: all clones were subsequently analyzed by flow cytometry. The 27Flip parental cell line was used as a negative control.
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pone-0003264-g003: Sustained OHT-independent flipping of the floxed cassette in 27Flip/Cre20 cells correlates with mer-Cre-mer expression level.A Following stable transfection of 27Flip cells with an mCrem expressing vector, 5 clones expressing various amounts of mCrem were selected. Cells from the clones were lysed and 50 µg of protein of each were run on an 8% SDS-PAGE gel. MCrem was visualized with polyclonal rabbit anti-Cre antibody (1∶2000, Novagen), with β-actin visualized with a polyclonal mouse antibody (1∶40000, Sigma) as a loading control. 27Flip/Cre15 and -20 were designated “high expressors”, and 27Flip/Cre26, -31, and -35 were designated “low expressors”. B 27Flip/Cre clones were either left untreated or treated with 1 µM OHT for 48 hours and then allowed to grow in culture for 18 days: all clones were subsequently analyzed by flow cytometry. The 27Flip parental cell line was used as a negative control.

Mentions: Several previous observations suggest that control of mCrem recombination is less than perfect [11], [20], [26], [27]. Based on these observations, we were interested in clarifying the role of mCrem expression level in OHT-independent recombination. We hypothesized that low frequency OHT-independent recombination by mCrem is mCrem intrinsic, and thus that OHT-independent Cre activity should be demonstrable in other mCrem expressing clones, and furthermore that as more mCrem would provide more activity, the level of constitutive flipping should correlate with mCrem expression level. To test this hypothesis, we re-transfected 27Flip cells with the mCrem containing vector to generate a panel of 27Flip/Cre clones expressing varying amounts of mCrem by western blot (Fig. 3A). These clones were assigned to either “high” (27Flip/Cre15 and -20) or “low” (27Flip/Cre26, -31, and -35) mCrem expressing categories and compared for flipping in the presence and absence of 1 µM OHT (Fig. 3B). Consistent with mCrem possessing intrinsic OHT-independent recombination activity, clones with high expression showed OHT-independent Cre activity, while low expressing clones exhibited little to no activity. Although the apparent decrease in flipping for the high expressing clones at high OHT concentrations suggested a potential difference in efficiency of recombination depending on the orientation of the cassette, repeating these experiments revealed variation in the size of the red population ranging from 38–60% that changed from day to day (data not shown), suggesting that background fluctuations in the population occur in the context of constitutive ongoing flipping. Importantly, all clones responded well to OHT treatment based on their establishing a ∼50∶50 ratio of red to white cells over a 48 hour OHT treatment (with a 50/50 ratio indicating flipping in ∼100% of cells). Note that clone 35, which at first glance appeared to have less red cells than other clones, showed poorer separation of red and white populations than other clones, making the percentage of white vs. red cells less precise in this clone than others.


Characterization of parameters required for effective use of tamoxifen-regulated recombination.

Buelow B, Scharenberg AM - PLoS ONE (2008)

Sustained OHT-independent flipping of the floxed cassette in 27Flip/Cre20 cells correlates with mer-Cre-mer expression level.A Following stable transfection of 27Flip cells with an mCrem expressing vector, 5 clones expressing various amounts of mCrem were selected. Cells from the clones were lysed and 50 µg of protein of each were run on an 8% SDS-PAGE gel. MCrem was visualized with polyclonal rabbit anti-Cre antibody (1∶2000, Novagen), with β-actin visualized with a polyclonal mouse antibody (1∶40000, Sigma) as a loading control. 27Flip/Cre15 and -20 were designated “high expressors”, and 27Flip/Cre26, -31, and -35 were designated “low expressors”. B 27Flip/Cre clones were either left untreated or treated with 1 µM OHT for 48 hours and then allowed to grow in culture for 18 days: all clones were subsequently analyzed by flow cytometry. The 27Flip parental cell line was used as a negative control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2533124&req=5

pone-0003264-g003: Sustained OHT-independent flipping of the floxed cassette in 27Flip/Cre20 cells correlates with mer-Cre-mer expression level.A Following stable transfection of 27Flip cells with an mCrem expressing vector, 5 clones expressing various amounts of mCrem were selected. Cells from the clones were lysed and 50 µg of protein of each were run on an 8% SDS-PAGE gel. MCrem was visualized with polyclonal rabbit anti-Cre antibody (1∶2000, Novagen), with β-actin visualized with a polyclonal mouse antibody (1∶40000, Sigma) as a loading control. 27Flip/Cre15 and -20 were designated “high expressors”, and 27Flip/Cre26, -31, and -35 were designated “low expressors”. B 27Flip/Cre clones were either left untreated or treated with 1 µM OHT for 48 hours and then allowed to grow in culture for 18 days: all clones were subsequently analyzed by flow cytometry. The 27Flip parental cell line was used as a negative control.
Mentions: Several previous observations suggest that control of mCrem recombination is less than perfect [11], [20], [26], [27]. Based on these observations, we were interested in clarifying the role of mCrem expression level in OHT-independent recombination. We hypothesized that low frequency OHT-independent recombination by mCrem is mCrem intrinsic, and thus that OHT-independent Cre activity should be demonstrable in other mCrem expressing clones, and furthermore that as more mCrem would provide more activity, the level of constitutive flipping should correlate with mCrem expression level. To test this hypothesis, we re-transfected 27Flip cells with the mCrem containing vector to generate a panel of 27Flip/Cre clones expressing varying amounts of mCrem by western blot (Fig. 3A). These clones were assigned to either “high” (27Flip/Cre15 and -20) or “low” (27Flip/Cre26, -31, and -35) mCrem expressing categories and compared for flipping in the presence and absence of 1 µM OHT (Fig. 3B). Consistent with mCrem possessing intrinsic OHT-independent recombination activity, clones with high expression showed OHT-independent Cre activity, while low expressing clones exhibited little to no activity. Although the apparent decrease in flipping for the high expressing clones at high OHT concentrations suggested a potential difference in efficiency of recombination depending on the orientation of the cassette, repeating these experiments revealed variation in the size of the red population ranging from 38–60% that changed from day to day (data not shown), suggesting that background fluctuations in the population occur in the context of constitutive ongoing flipping. Importantly, all clones responded well to OHT treatment based on their establishing a ∼50∶50 ratio of red to white cells over a 48 hour OHT treatment (with a 50/50 ratio indicating flipping in ∼100% of cells). Note that clone 35, which at first glance appeared to have less red cells than other clones, showed poorer separation of red and white populations than other clones, making the percentage of white vs. red cells less precise in this clone than others.

Bottom Line: In the DT40 chicken B cell line, we generated an mCrem-based reversible switch for conditional regulation of a transgene, and in contrast with previous work, observed significant constitutive activity of mCrem.This prompted us to use our system for analysis of the parameters governing tamoxifen-regulated mCrem recombination of a genomic target.We also observe time and dose-dependent effects on mCrem activity which suggest limitations on the use of conditional targeting approaches for applications which require tight temporal coordination of Cre action within a cell population.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Washington, Seattle Children's Hospital Research Institute, Seattle, Washington, USA.

ABSTRACT
Conditional gene targeting using the Cre-loxp system is a well established technique in numerous in vitro and in vivo systems. Ligand regulated forms of Cre have been increasingly used in these applications in order to gain temporal and spatial control over conditional targeting. The tamoxifen-regulated Cre variant mer-Cre-mer (mCrem) is widely utilized because of its reputation for tight regulation in the absence of its tamoxifen ligand. In the DT40 chicken B cell line, we generated an mCrem-based reversible switch for conditional regulation of a transgene, and in contrast with previous work, observed significant constitutive activity of mCrem. This prompted us to use our system for analysis of the parameters governing tamoxifen-regulated mCrem recombination of a genomic target. We find that robust mCrem expression correlates with a high level of tamoxifen-independent Cre activity, while clones expressing mCrem at the limit of western blot detection exhibit extremely tight regulation. We also observe time and dose-dependent effects on mCrem activity which suggest limitations on the use of conditional targeting approaches for applications which require tight temporal coordination of Cre action within a cell population.

Show MeSH
Related in: MedlinePlus