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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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(A) Indirect IF of topo 2α and topo 2β in the parental, αKD and αβKD cell lines after 6 days dox exposure. Cells were cytospun onto slides. Topo 2α was detected using anti-Flag antibody (Texas Red) and topo 2β using anti-GFP (FITC). DNA was counterstained using DAPI (blue). Scale bar, 10 μm. (B) Western blots showing robust knockdown, over 4 days dox exposure, of topo 2α in αKD cells and of both isoforms in αβKD cells. Topo 2α was detected using anti-Flag antibody and 2β using anti-GFP. β-actin was used as a loading control. (C) Growth curves of the parental (blue), αKD (red) and αβKD (green) cell lines grown in the presence (closed circles) and absence (closed squares) of 2.5 μg ml–1 dox for 6 days. Data points represent the mean (±SD) based on ≥3 independent experiments.
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Figure 4: (A) Indirect IF of topo 2α and topo 2β in the parental, αKD and αβKD cell lines after 6 days dox exposure. Cells were cytospun onto slides. Topo 2α was detected using anti-Flag antibody (Texas Red) and topo 2β using anti-GFP (FITC). DNA was counterstained using DAPI (blue). Scale bar, 10 μm. (B) Western blots showing robust knockdown, over 4 days dox exposure, of topo 2α in αKD cells and of both isoforms in αβKD cells. Topo 2α was detected using anti-Flag antibody and 2β using anti-GFP. β-actin was used as a loading control. (C) Growth curves of the parental (blue), αKD (red) and αβKD (green) cell lines grown in the presence (closed circles) and absence (closed squares) of 2.5 μg ml–1 dox for 6 days. Data points represent the mean (±SD) based on ≥3 independent experiments.

Mentions: One line displaying robust topo 2α depletion (KD clone 8, referred to subsequently as αKD) was re-transfected, using a shtopo 2β expression construct (Figure 2D) conferring zeocin-resistance. After an initial screen of several transfectants, one line showing robust topo 2β depletion was characterized further, with the KD of both isoforms being confirmed by indirect IF of fixed cells (Figure 4A) and western blotting (Figure 4B) (cell line designated αβKD).


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)

(A) Indirect IF of topo 2α and topo 2β in the parental, αKD and αβKD cell lines after 6 days dox exposure. Cells were cytospun onto slides. Topo 2α was detected using anti-Flag antibody (Texas Red) and topo 2β using anti-GFP (FITC). DNA was counterstained using DAPI (blue). Scale bar, 10 μm. (B) Western blots showing robust knockdown, over 4 days dox exposure, of topo 2α in αKD cells and of both isoforms in αβKD cells. Topo 2α was detected using anti-Flag antibody and 2β using anti-GFP. β-actin was used as a loading control. (C) Growth curves of the parental (blue), αKD (red) and αβKD (green) cell lines grown in the presence (closed circles) and absence (closed squares) of 2.5 μg ml–1 dox for 6 days. Data points represent the mean (±SD) based on ≥3 independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: (A) Indirect IF of topo 2α and topo 2β in the parental, αKD and αβKD cell lines after 6 days dox exposure. Cells were cytospun onto slides. Topo 2α was detected using anti-Flag antibody (Texas Red) and topo 2β using anti-GFP (FITC). DNA was counterstained using DAPI (blue). Scale bar, 10 μm. (B) Western blots showing robust knockdown, over 4 days dox exposure, of topo 2α in αKD cells and of both isoforms in αβKD cells. Topo 2α was detected using anti-Flag antibody and 2β using anti-GFP. β-actin was used as a loading control. (C) Growth curves of the parental (blue), αKD (red) and αβKD (green) cell lines grown in the presence (closed circles) and absence (closed squares) of 2.5 μg ml–1 dox for 6 days. Data points represent the mean (±SD) based on ≥3 independent experiments.
Mentions: One line displaying robust topo 2α depletion (KD clone 8, referred to subsequently as αKD) was re-transfected, using a shtopo 2β expression construct (Figure 2D) conferring zeocin-resistance. After an initial screen of several transfectants, one line showing robust topo 2β depletion was characterized further, with the KD of both isoforms being confirmed by indirect IF of fixed cells (Figure 4A) and western blotting (Figure 4B) (cell line designated αβKD).

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