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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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Efficiency of OHT-dependent flipping of the floxed cassette correlates with duration of OHT exposure in low mCrem expressors.A 27Flip/Cre clones were either left untreated or treated with 1 nM OHT for 24, 10, 5, 2, or 1 hours, and then allowed to grow in culture for 5 days: subsequently all clones were analyzed by flow cytometry. 27Flip parental cells were used as a negative control. B 27Flip/Cre31 cells were either left untreated or treated with 100 nM OHT for 1, 2, 5, 10, 30, 60, 120, or 240 minutes and then allowed to grow in culture for 5 days: subsequently all clones were analyzed by flow cytometry. X-axis, mCherry fluorescence, Y-axis, cell number.
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pone-0003264-g005: Efficiency of OHT-dependent flipping of the floxed cassette correlates with duration of OHT exposure in low mCrem expressors.A 27Flip/Cre clones were either left untreated or treated with 1 nM OHT for 24, 10, 5, 2, or 1 hours, and then allowed to grow in culture for 5 days: subsequently all clones were analyzed by flow cytometry. 27Flip parental cells were used as a negative control. B 27Flip/Cre31 cells were either left untreated or treated with 100 nM OHT for 1, 2, 5, 10, 30, 60, 120, or 240 minutes and then allowed to grow in culture for 5 days: subsequently all clones were analyzed by flow cytometry. X-axis, mCherry fluorescence, Y-axis, cell number.

Mentions: We noted that at all concentrations of OHT, the majority of treated cells became positive for red fluorescence within 24 hours (data not shown), suggesting that the majority of cells may already have initiated switching well before the 48 hours of OHT exposure typically employed by our and other labs [24]. We therefore set out to identify the effect of varying OHT exposure time in clones expressing different amounts of mCrem. We treated the 27Flip/Cre clones with 1 nM OHT for 1, 2, 5, 10, and 24 hours before removing OHT and allowing the cells to grow for five days in culture, at which point we examined the cells for red fluorescence by FACS (Fig. 5A). In all clones except the high expressor 27Flip/Cre15, a steady decline in recombination activity was evident as OHT exposure time decreased: for 27Flip/Cre15 flipping was already at ∼100% in the untreated population. Coupled with the dose response of the various clones, these results also provided further support for our conclusion from Fig. 3 that recombination efficiency decreases with decreasing mCrem expression. Flipping in 100% of the cells in all clones except 27Flip/Cre15 was not seen at exposure times less than 48 hours.


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

Buelow B, Scharenberg AM - PLoS ONE (2008)

Efficiency of OHT-dependent flipping of the floxed cassette correlates with duration of OHT exposure in low mCrem expressors.A 27Flip/Cre clones were either left untreated or treated with 1 nM OHT for 24, 10, 5, 2, or 1 hours, and then allowed to grow in culture for 5 days: subsequently all clones were analyzed by flow cytometry. 27Flip parental cells were used as a negative control. B 27Flip/Cre31 cells were either left untreated or treated with 100 nM OHT for 1, 2, 5, 10, 30, 60, 120, or 240 minutes and then allowed to grow in culture for 5 days: subsequently all clones were analyzed by flow cytometry. 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-g005: Efficiency of OHT-dependent flipping of the floxed cassette correlates with duration of OHT exposure in low mCrem expressors.A 27Flip/Cre clones were either left untreated or treated with 1 nM OHT for 24, 10, 5, 2, or 1 hours, and then allowed to grow in culture for 5 days: subsequently all clones were analyzed by flow cytometry. 27Flip parental cells were used as a negative control. B 27Flip/Cre31 cells were either left untreated or treated with 100 nM OHT for 1, 2, 5, 10, 30, 60, 120, or 240 minutes and then allowed to grow in culture for 5 days: subsequently all clones were analyzed by flow cytometry. X-axis, mCherry fluorescence, Y-axis, cell number.
Mentions: We noted that at all concentrations of OHT, the majority of treated cells became positive for red fluorescence within 24 hours (data not shown), suggesting that the majority of cells may already have initiated switching well before the 48 hours of OHT exposure typically employed by our and other labs [24]. We therefore set out to identify the effect of varying OHT exposure time in clones expressing different amounts of mCrem. We treated the 27Flip/Cre clones with 1 nM OHT for 1, 2, 5, 10, and 24 hours before removing OHT and allowing the cells to grow for five days in culture, at which point we examined the cells for red fluorescence by FACS (Fig. 5A). In all clones except the high expressor 27Flip/Cre15, a steady decline in recombination activity was evident as OHT exposure time decreased: for 27Flip/Cre15 flipping was already at ∼100% in the untreated population. Coupled with the dose response of the various clones, these results also provided further support for our conclusion from Fig. 3 that recombination efficiency decreases with decreasing mCrem expression. Flipping in 100% of the cells in all clones except 27Flip/Cre15 was not seen at exposure times less than 48 hours.

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