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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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Related in: MedlinePlus

Sustained OHT-independent flipping of the floxed cassette in 27Flip/Cre20 cells is a stable, ongoing process.mCherry (+) and mCherry (−) 27Flip/Cre20 cells were separated by FACS and grown in culture for ∼7 weeks. During this time period, a steady decrease in the purity of the sorted populations was observed by flow cytometry. X-axis, mCherry fluorescence, Y-axis, cell number.
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pone-0003264-g002: Sustained OHT-independent flipping of the floxed cassette in 27Flip/Cre20 cells is a stable, ongoing process.mCherry (+) and mCherry (−) 27Flip/Cre20 cells were separated by FACS and grown in culture for ∼7 weeks. During this time period, a steady decrease in the purity of the sorted populations was observed by flow cytometry. X-axis, mCherry fluorescence, Y-axis, cell number.

Mentions: We initially hypothesized that electroporation or other stressors associated with transduction of DT40 cells with the mCrem containing construct might lead to inefficient exclusion of mCrem from the nucleus and subsequent Cre dependent switching of our construct in affected cells. To test this hypothesis, we sorted mCherry negative (“white”) and mCherry-positive (“red”) cells by flow cytometry, and monitored the fluorescence of the separated populations over time in the absence of OHT (Fig. 2). If mCrem activity were a transient event associated with electroporation, these sorted populations would be expected to remain stable over time. However, we observed that white cells showed an outgrowth of red cells and vice versa over a course of 7 weeks in culture. Interestingly, red cells converted to white cells more rapidly, a phenomenon potentially related to idiosyncratic differences in susceptibility of the flip cassette to recombination, or a survival disadvantage associated with loss of NUDT9 expression. These results suggested that in contrast to previous results, mCrem was capable of mediating significant levels of constitutive recombination activity. Because such unlicensed mCrem dependent recombination is a potential confounding factor for the reversible switching approach to conditional gene inactivation, we set out to better characterize this phenomenon.


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 is a stable, ongoing process.mCherry (+) and mCherry (−) 27Flip/Cre20 cells were separated by FACS and grown in culture for ∼7 weeks. During this time period, a steady decrease in the purity of the sorted populations was observed 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-g002: Sustained OHT-independent flipping of the floxed cassette in 27Flip/Cre20 cells is a stable, ongoing process.mCherry (+) and mCherry (−) 27Flip/Cre20 cells were separated by FACS and grown in culture for ∼7 weeks. During this time period, a steady decrease in the purity of the sorted populations was observed by flow cytometry. X-axis, mCherry fluorescence, Y-axis, cell number.
Mentions: We initially hypothesized that electroporation or other stressors associated with transduction of DT40 cells with the mCrem containing construct might lead to inefficient exclusion of mCrem from the nucleus and subsequent Cre dependent switching of our construct in affected cells. To test this hypothesis, we sorted mCherry negative (“white”) and mCherry-positive (“red”) cells by flow cytometry, and monitored the fluorescence of the separated populations over time in the absence of OHT (Fig. 2). If mCrem activity were a transient event associated with electroporation, these sorted populations would be expected to remain stable over time. However, we observed that white cells showed an outgrowth of red cells and vice versa over a course of 7 weeks in culture. Interestingly, red cells converted to white cells more rapidly, a phenomenon potentially related to idiosyncratic differences in susceptibility of the flip cassette to recombination, or a survival disadvantage associated with loss of NUDT9 expression. These results suggested that in contrast to previous results, mCrem was capable of mediating significant levels of constitutive recombination activity. Because such unlicensed mCrem dependent recombination is a potential confounding factor for the reversible switching approach to conditional gene inactivation, we set out to better characterize this phenomenon.

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