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

Buelow B, Scharenberg AM - PLoS ONE (2008)

Bottom Line: Conditional gene targeting using the Cre-loxp system is a well established technique in numerous in vitro and in vivo systems.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.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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Related in: MedlinePlus

OHT-dependent flipping of the floxed cassette saturates by 10 nM OHT.27Flip/Cre clones were either left untreated or treated with 1, 10, or 100 nM OHT for 48 hours and then allowed to grow in culture for 5 days from the onset of OHT treatment: subsequently all clones were analyzed by flow cytometry. 27Flip parental cells were used as a negative control. X-axis, mCherry fluorescence, Y-axis, cell number.
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pone-0003264-g004: OHT-dependent flipping of the floxed cassette saturates by 10 nM OHT.27Flip/Cre clones were either left untreated or treated with 1, 10, or 100 nM OHT for 48 hours and then allowed to grow in culture for 5 days from the onset of OHT treatment: subsequently all clones were analyzed by flow cytometry. 27Flip parental cells were used as a negative control. X-axis, mCherry fluorescence, Y-axis, cell number.

Mentions: In a previous study [24] an OHT concentration of 50 nM was used to induce mCrem mediated recombination, significantly lower than the 1 µM used in the studies above. Thus, we wanted to investigate the interaction between mCrem expression level and OHT dose for induction of mCrem mediated recombination. Thus, we treated both the high and low expressing 27Flip/Cre clones with 100, 10, or 1 nM OHT for 48 hours, and compared the treatment groups for Cre activity at 5 days after the start of OHT treatment (Fig. 4). Since during OHT treatment the construct spends equal time in the forward and reverse orientations, all cells express mCherry during this time: as such, we included a “rest” period of 3+ days to allow white cells to lose any residual fluorescence. Clones 15 and 20 with constitutive flipping activity exhibited only minor changes in population distribution after OHT treatment - these minor changes may reflect differing efficiencies of flipping in the forward and reverse directions or differential effects of tamoxifen toxicity on the two populations at the higher doses. Clones with lower mCrem expression all exhibited dose dependent recombination, with the clone with the lowest apparent mCrem expression (based on a qualitative assessment of the western blot in Fig. 3A), 27Flip/Cre31, showing the lowest level of activity at 1 nM Cre. This result suggests that at sufficiently low OHT concentration, mCrem expression level becomes limiting for recombination efficiency. Nevertheless, although mCrem expression in this clone was near the limit of detection in our western blot, we saw flipping in 100% of cells at 10 nM OHT, suggesting that this concentration is sufficient to maximize Cre activity when applied for 48 hours. Consistent with previous work [11] we observed significant mCrem mediated toxicity at 1 µM and 100 nM OHT, which decreased at 10 nM and was essentially absent at 1 nM. In addition, the frequency of cell death was consistently higher among high mCrem expressors as compared to low expressors at 100 and 10 nm OHT, plateauing at 1 µM and being undetectable at 1 nM for all clones (data not shown).


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

Buelow B, Scharenberg AM - PLoS ONE (2008)

OHT-dependent flipping of the floxed cassette saturates by 10 nM OHT.27Flip/Cre clones were either left untreated or treated with 1, 10, or 100 nM OHT for 48 hours and then allowed to grow in culture for 5 days from the onset of OHT treatment: subsequently all clones were analyzed by flow cytometry. 27Flip parental cells were used as a negative control. X-axis, mCherry fluorescence, Y-axis, cell number.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2533124&req=5

pone-0003264-g004: OHT-dependent flipping of the floxed cassette saturates by 10 nM OHT.27Flip/Cre clones were either left untreated or treated with 1, 10, or 100 nM OHT for 48 hours and then allowed to grow in culture for 5 days from the onset of OHT treatment: subsequently all clones were analyzed by flow cytometry. 27Flip parental cells were used as a negative control. X-axis, mCherry fluorescence, Y-axis, cell number.
Mentions: In a previous study [24] an OHT concentration of 50 nM was used to induce mCrem mediated recombination, significantly lower than the 1 µM used in the studies above. Thus, we wanted to investigate the interaction between mCrem expression level and OHT dose for induction of mCrem mediated recombination. Thus, we treated both the high and low expressing 27Flip/Cre clones with 100, 10, or 1 nM OHT for 48 hours, and compared the treatment groups for Cre activity at 5 days after the start of OHT treatment (Fig. 4). Since during OHT treatment the construct spends equal time in the forward and reverse orientations, all cells express mCherry during this time: as such, we included a “rest” period of 3+ days to allow white cells to lose any residual fluorescence. Clones 15 and 20 with constitutive flipping activity exhibited only minor changes in population distribution after OHT treatment - these minor changes may reflect differing efficiencies of flipping in the forward and reverse directions or differential effects of tamoxifen toxicity on the two populations at the higher doses. Clones with lower mCrem expression all exhibited dose dependent recombination, with the clone with the lowest apparent mCrem expression (based on a qualitative assessment of the western blot in Fig. 3A), 27Flip/Cre31, showing the lowest level of activity at 1 nM Cre. This result suggests that at sufficiently low OHT concentration, mCrem expression level becomes limiting for recombination efficiency. Nevertheless, although mCrem expression in this clone was near the limit of detection in our western blot, we saw flipping in 100% of cells at 10 nM OHT, suggesting that this concentration is sufficient to maximize Cre activity when applied for 48 hours. Consistent with previous work [11] we observed significant mCrem mediated toxicity at 1 µM and 100 nM OHT, which decreased at 10 nM and was essentially absent at 1 nM. In addition, the frequency of cell death was consistently higher among high mCrem expressors as compared to low expressors at 100 and 10 nm OHT, plateauing at 1 µM and being undetectable at 1 nM for all clones (data not shown).

Bottom Line: Conditional gene targeting using the Cre-loxp system is a well established technique in numerous in vitro and in vivo systems.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.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