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Studying vertebrate topoisomerase 2 function using a conditional knockdown system in DT40 cells.

Johnson M, Phua HH, Bennett SC, Spence JM, Farr CJ - Nucleic Acids Res. (2009)

Bottom Line: We have generated and characterized stable DT40 transfectants in which both topo 2 genes have been in situ tagged using gene targeting, and from which the mRNA of both topoisomerase 2 isoforms can be conditionally depleted through the tetracycline-induced expression of short hairpin RNAs.The cell cycle phenotype of topo 2-depleted DT40 cells has been compared with that previously reported for other vertebrate cells depleted either of topo 2alpha through gene targeting, or depleted of both isoforms simultaneously by transient RNAi.In addition, the DT40 knockdown system has been used to explore whether excess catenation arising through topo 2 depletion is sufficient to trigger the G2 catenation (or decatenation) checkpoint, proposed to exist in differentiated vertebrate cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Downing St, Cambridge CB2 3EH, UK.

ABSTRACT
DT40 is a B-cell lymphoma-derived avian cell line widely used to study cell autonomous gene function because of the high rates with which DNA constructs are homologously recombined into its genome. Here, we demonstrate that the power of the DT40 system can be extended yet further through the use of RNA interference as an alternative to gene targeting. We have generated and characterized stable DT40 transfectants in which both topo 2 genes have been in situ tagged using gene targeting, and from which the mRNA of both topoisomerase 2 isoforms can be conditionally depleted through the tetracycline-induced expression of short hairpin RNAs. The cell cycle phenotype of topo 2-depleted DT40 cells has been compared with that previously reported for other vertebrate cells depleted either of topo 2alpha through gene targeting, or depleted of both isoforms simultaneously by transient RNAi. In addition, the DT40 knockdown system has been used to explore whether excess catenation arising through topo 2 depletion is sufficient to trigger the G2 catenation (or decatenation) checkpoint, proposed to exist in differentiated vertebrate cells.

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Summary graph of the accumulation of cells staining positive for the mitotic marker pS10H3 (quantified by flow cytometry) after 8 h in the presence of nocodazole, in cultures treated with dox for 0 or 3 days (based on nine independent experiments) (white bars). The impact of 1 μM ICRF-193 (present throughout the 8-h period with nocodazole) on progression into M phase is shown alongside (three independent experiments) (grey bars). To address whether topo 2 depletion has any effect on the ability of DT40 cells to arrest in G2 in response to DSBs, cultures were exposed to 4 Gy of X-irradiation immediately prior to nocodazole addition (three independent experiments) (black bars). Values correspond to the means (±SD).
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Figure 8: Summary graph of the accumulation of cells staining positive for the mitotic marker pS10H3 (quantified by flow cytometry) after 8 h in the presence of nocodazole, in cultures treated with dox for 0 or 3 days (based on nine independent experiments) (white bars). The impact of 1 μM ICRF-193 (present throughout the 8-h period with nocodazole) on progression into M phase is shown alongside (three independent experiments) (grey bars). To address whether topo 2 depletion has any effect on the ability of DT40 cells to arrest in G2 in response to DSBs, cultures were exposed to 4 Gy of X-irradiation immediately prior to nocodazole addition (three independent experiments) (black bars). Values correspond to the means (±SD).

Mentions: A two-tailed t-test (unpaired) was used to assess the statistical significance of the observed differences in cell-cycle data in Figures 6 and 8.Figure 1.


Studying vertebrate topoisomerase 2 function using a conditional knockdown system in DT40 cells.

Johnson M, Phua HH, Bennett SC, Spence JM, Farr CJ - Nucleic Acids Res. (2009)

Summary graph of the accumulation of cells staining positive for the mitotic marker pS10H3 (quantified by flow cytometry) after 8 h in the presence of nocodazole, in cultures treated with dox for 0 or 3 days (based on nine independent experiments) (white bars). The impact of 1 μM ICRF-193 (present throughout the 8-h period with nocodazole) on progression into M phase is shown alongside (three independent experiments) (grey bars). To address whether topo 2 depletion has any effect on the ability of DT40 cells to arrest in G2 in response to DSBs, cultures were exposed to 4 Gy of X-irradiation immediately prior to nocodazole addition (three independent experiments) (black bars). Values correspond to the means (±SD).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724289&req=5

Figure 8: Summary graph of the accumulation of cells staining positive for the mitotic marker pS10H3 (quantified by flow cytometry) after 8 h in the presence of nocodazole, in cultures treated with dox for 0 or 3 days (based on nine independent experiments) (white bars). The impact of 1 μM ICRF-193 (present throughout the 8-h period with nocodazole) on progression into M phase is shown alongside (three independent experiments) (grey bars). To address whether topo 2 depletion has any effect on the ability of DT40 cells to arrest in G2 in response to DSBs, cultures were exposed to 4 Gy of X-irradiation immediately prior to nocodazole addition (three independent experiments) (black bars). Values correspond to the means (±SD).
Mentions: A two-tailed t-test (unpaired) was used to assess the statistical significance of the observed differences in cell-cycle data in Figures 6 and 8.Figure 1.

Bottom Line: We have generated and characterized stable DT40 transfectants in which both topo 2 genes have been in situ tagged using gene targeting, and from which the mRNA of both topoisomerase 2 isoforms can be conditionally depleted through the tetracycline-induced expression of short hairpin RNAs.The cell cycle phenotype of topo 2-depleted DT40 cells has been compared with that previously reported for other vertebrate cells depleted either of topo 2alpha through gene targeting, or depleted of both isoforms simultaneously by transient RNAi.In addition, the DT40 knockdown system has been used to explore whether excess catenation arising through topo 2 depletion is sufficient to trigger the G2 catenation (or decatenation) checkpoint, proposed to exist in differentiated vertebrate cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Downing St, Cambridge CB2 3EH, UK.

ABSTRACT
DT40 is a B-cell lymphoma-derived avian cell line widely used to study cell autonomous gene function because of the high rates with which DNA constructs are homologously recombined into its genome. Here, we demonstrate that the power of the DT40 system can be extended yet further through the use of RNA interference as an alternative to gene targeting. We have generated and characterized stable DT40 transfectants in which both topo 2 genes have been in situ tagged using gene targeting, and from which the mRNA of both topoisomerase 2 isoforms can be conditionally depleted through the tetracycline-induced expression of short hairpin RNAs. The cell cycle phenotype of topo 2-depleted DT40 cells has been compared with that previously reported for other vertebrate cells depleted either of topo 2alpha through gene targeting, or depleted of both isoforms simultaneously by transient RNAi. In addition, the DT40 knockdown system has been used to explore whether excess catenation arising through topo 2 depletion is sufficient to trigger the G2 catenation (or decatenation) checkpoint, proposed to exist in differentiated vertebrate cells.

Show MeSH
Related in: MedlinePlus