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Dissecting mitosis by RNAi in Drosophila tissue culture cells.

Maiato H, Sunkel CE, Earnshaw WC - Biol Proced Online (2003)

Bottom Line: With the advent of whole genome sequencing it is expected that RNAi-based screenings will be one method of choice for the identification and study of novel genes involved in particular cellular processes.In this paper we focused particularly on the procedures for the proper phenotypic analysis of cells after RNAi-mediated depletion of proteins required for mitosis, the process by which the genetic information is segregated equally between daughter cells.We use RNAi of the microtubule-associated protein MAST/Orbit as an example for the usefulness of the technique.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratório de Genética Molecular, Instituto de Biologia Molecular e Celular, Universidade do Porto. Rua Campo Alegre, 823, 4150-180 Porto. Portugal.

ABSTRACT
Here we describe a detailed methodology to study the function of genes whose products function during mitosis by dsRNA-mediated interference (RNAi) in cultured cells of Drosophila melanogaster. This procedure is particularly useful for the analysis of genes for which genetic mutations are not available or for the dissection of complicated phenotypes derived from the analysis of such mutants. With the advent of whole genome sequencing it is expected that RNAi-based screenings will be one method of choice for the identification and study of novel genes involved in particular cellular processes. In this paper we focused particularly on the procedures for the proper phenotypic analysis of cells after RNAi-mediated depletion of proteins required for mitosis, the process by which the genetic information is segregated equally between daughter cells. We use RNAi of the microtubule-associated protein MAST/Orbit as an example for the usefulness of the technique.

No MeSH data available.


Related in: MedlinePlus

Immunofluorescence analysis of mitotic S2 cells after RNAi. Chromosomes were stained with DAPI (blue), microtubules were stained with an anti-α-tubulin antibody (green), centrosomes were stained with an anti-CP-190 antibody and the centromeres were stained with an anti-CID antibody (both white or red in the merged images). (A, A’ and E, E’) Control metaphase cells showing a well organized bipolar spindle with one centrosome at each pole and with the chromosomes correctly aligned at the metaphase plate. (B, B’ and F, F’) 72 h after RNAi, cells form monopolar spindles with the centrosomes clustered in the centre and the centromeres dispersed in the monoaster. (C, C’) Polyploid cell 96 h after RNAi showing multiple centrosomes. (G, G’) Cell showing abnormal chromosome congression 72 h after RNAi. (D, D’) Abnormal anaphase-like cells 120 h after RNAi showing a bipolar spindle where the centrosomes are clustered at only one of the poles. (E, E’) Another anaphase-like cell where non-disjoined chromosomes have attempted to segregate towards opposite poles. Scale bar is 5 µm.
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Figure 2: Immunofluorescence analysis of mitotic S2 cells after RNAi. Chromosomes were stained with DAPI (blue), microtubules were stained with an anti-α-tubulin antibody (green), centrosomes were stained with an anti-CP-190 antibody and the centromeres were stained with an anti-CID antibody (both white or red in the merged images). (A, A’ and E, E’) Control metaphase cells showing a well organized bipolar spindle with one centrosome at each pole and with the chromosomes correctly aligned at the metaphase plate. (B, B’ and F, F’) 72 h after RNAi, cells form monopolar spindles with the centrosomes clustered in the centre and the centromeres dispersed in the monoaster. (C, C’) Polyploid cell 96 h after RNAi showing multiple centrosomes. (G, G’) Cell showing abnormal chromosome congression 72 h after RNAi. (D, D’) Abnormal anaphase-like cells 120 h after RNAi showing a bipolar spindle where the centrosomes are clustered at only one of the poles. (E, E’) Another anaphase-like cell where non-disjoined chromosomes have attempted to segregate towards opposite poles. Scale bar is 5 µm.

Mentions: Detection of the initial phenotype resulting from addition of dsRNA to the cell culture is an important goal of the RNAi experiment. However, this can often be very tricky, especially for proteins that have multiple roles in a cellular process, as is often the case for regulatory proteins such as kinases or phosphatases. Ideally, in order to minimize heterogeneity between different cell populations, a small aliquot of cells from each time point used for immunoblotting should be used for immunofluorescence analysis. Nevertheless, this problem can be minimized by collecting, from a parallel experiment, samples at several time points after addition of the dsRNA to the culture followed by analysis by immunofluorescence using appropriate antibodies (again, samples must be collected from both RNAi and control experiments at each time point). For this purpose we repeated the same procedures described for the immunoblot, but using LAB-TEK permanox 2-chamber slides from Nalge Nunc (distributed by Gibco). Due to the size of the chambers, we used 5 x 105 cells/0.5 ml of media/chamber, and added half the amount of dsRNA used for the immunoblot experiment (in this case 15 µg/chamber), with one chamber used for the experimental and the other for the corresponding control. After serum starvation for 1 h, 1 ml of Schneider’s Drosophila medium supplemented with FBS was added to each chamber and the slides were returned to the incubator at 25°C. As S2 cells grow in suspension it was necessary at each time point to centrifuge cells onto the slides for 15 min at 4,000 rpm at room temperature to render them adherent. Alternatively, the cells could be grown in 6 well plates as described for the immunoblot, and at each time point cytospun or left to adhere for 2 hours onto sterile poly-L-lysine treated slides (BDH). Cells were then immediately fixed and permeabilized and processed for immunofluorescence. Alternatively, after fixation, cells can be kept in PBS at 4°C up to one week and the complete set of cells from each time point processed at the same time for immunofluorescence (Fig. 2). In the case of MAST RNAi, the first visible abnormality that we observed was an increase in the mitotic index caused by the accumulation of cells in a prometaphase-like stage. Among these prometaphase cells, we observed two distinct populations, those with monopolar spindles (Fig. 2B and 2F) and those in which the spindle was bipolar but on which the chromosomes could not align at a metaphase plate (Fig. 2G). We defined these as the primary consequences due to MAST depletion. These results were confirmed independently by in vivo analysis of mast mutant embryos (17).


Dissecting mitosis by RNAi in Drosophila tissue culture cells.

Maiato H, Sunkel CE, Earnshaw WC - Biol Proced Online (2003)

Immunofluorescence analysis of mitotic S2 cells after RNAi. Chromosomes were stained with DAPI (blue), microtubules were stained with an anti-α-tubulin antibody (green), centrosomes were stained with an anti-CP-190 antibody and the centromeres were stained with an anti-CID antibody (both white or red in the merged images). (A, A’ and E, E’) Control metaphase cells showing a well organized bipolar spindle with one centrosome at each pole and with the chromosomes correctly aligned at the metaphase plate. (B, B’ and F, F’) 72 h after RNAi, cells form monopolar spindles with the centrosomes clustered in the centre and the centromeres dispersed in the monoaster. (C, C’) Polyploid cell 96 h after RNAi showing multiple centrosomes. (G, G’) Cell showing abnormal chromosome congression 72 h after RNAi. (D, D’) Abnormal anaphase-like cells 120 h after RNAi showing a bipolar spindle where the centrosomes are clustered at only one of the poles. (E, E’) Another anaphase-like cell where non-disjoined chromosomes have attempted to segregate towards opposite poles. Scale bar is 5 µm.
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Related In: Results  -  Collection

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Figure 2: Immunofluorescence analysis of mitotic S2 cells after RNAi. Chromosomes were stained with DAPI (blue), microtubules were stained with an anti-α-tubulin antibody (green), centrosomes were stained with an anti-CP-190 antibody and the centromeres were stained with an anti-CID antibody (both white or red in the merged images). (A, A’ and E, E’) Control metaphase cells showing a well organized bipolar spindle with one centrosome at each pole and with the chromosomes correctly aligned at the metaphase plate. (B, B’ and F, F’) 72 h after RNAi, cells form monopolar spindles with the centrosomes clustered in the centre and the centromeres dispersed in the monoaster. (C, C’) Polyploid cell 96 h after RNAi showing multiple centrosomes. (G, G’) Cell showing abnormal chromosome congression 72 h after RNAi. (D, D’) Abnormal anaphase-like cells 120 h after RNAi showing a bipolar spindle where the centrosomes are clustered at only one of the poles. (E, E’) Another anaphase-like cell where non-disjoined chromosomes have attempted to segregate towards opposite poles. Scale bar is 5 µm.
Mentions: Detection of the initial phenotype resulting from addition of dsRNA to the cell culture is an important goal of the RNAi experiment. However, this can often be very tricky, especially for proteins that have multiple roles in a cellular process, as is often the case for regulatory proteins such as kinases or phosphatases. Ideally, in order to minimize heterogeneity between different cell populations, a small aliquot of cells from each time point used for immunoblotting should be used for immunofluorescence analysis. Nevertheless, this problem can be minimized by collecting, from a parallel experiment, samples at several time points after addition of the dsRNA to the culture followed by analysis by immunofluorescence using appropriate antibodies (again, samples must be collected from both RNAi and control experiments at each time point). For this purpose we repeated the same procedures described for the immunoblot, but using LAB-TEK permanox 2-chamber slides from Nalge Nunc (distributed by Gibco). Due to the size of the chambers, we used 5 x 105 cells/0.5 ml of media/chamber, and added half the amount of dsRNA used for the immunoblot experiment (in this case 15 µg/chamber), with one chamber used for the experimental and the other for the corresponding control. After serum starvation for 1 h, 1 ml of Schneider’s Drosophila medium supplemented with FBS was added to each chamber and the slides were returned to the incubator at 25°C. As S2 cells grow in suspension it was necessary at each time point to centrifuge cells onto the slides for 15 min at 4,000 rpm at room temperature to render them adherent. Alternatively, the cells could be grown in 6 well plates as described for the immunoblot, and at each time point cytospun or left to adhere for 2 hours onto sterile poly-L-lysine treated slides (BDH). Cells were then immediately fixed and permeabilized and processed for immunofluorescence. Alternatively, after fixation, cells can be kept in PBS at 4°C up to one week and the complete set of cells from each time point processed at the same time for immunofluorescence (Fig. 2). In the case of MAST RNAi, the first visible abnormality that we observed was an increase in the mitotic index caused by the accumulation of cells in a prometaphase-like stage. Among these prometaphase cells, we observed two distinct populations, those with monopolar spindles (Fig. 2B and 2F) and those in which the spindle was bipolar but on which the chromosomes could not align at a metaphase plate (Fig. 2G). We defined these as the primary consequences due to MAST depletion. These results were confirmed independently by in vivo analysis of mast mutant embryos (17).

Bottom Line: With the advent of whole genome sequencing it is expected that RNAi-based screenings will be one method of choice for the identification and study of novel genes involved in particular cellular processes.In this paper we focused particularly on the procedures for the proper phenotypic analysis of cells after RNAi-mediated depletion of proteins required for mitosis, the process by which the genetic information is segregated equally between daughter cells.We use RNAi of the microtubule-associated protein MAST/Orbit as an example for the usefulness of the technique.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratório de Genética Molecular, Instituto de Biologia Molecular e Celular, Universidade do Porto. Rua Campo Alegre, 823, 4150-180 Porto. Portugal.

ABSTRACT
Here we describe a detailed methodology to study the function of genes whose products function during mitosis by dsRNA-mediated interference (RNAi) in cultured cells of Drosophila melanogaster. This procedure is particularly useful for the analysis of genes for which genetic mutations are not available or for the dissection of complicated phenotypes derived from the analysis of such mutants. With the advent of whole genome sequencing it is expected that RNAi-based screenings will be one method of choice for the identification and study of novel genes involved in particular cellular processes. In this paper we focused particularly on the procedures for the proper phenotypic analysis of cells after RNAi-mediated depletion of proteins required for mitosis, the process by which the genetic information is segregated equally between daughter cells. We use RNAi of the microtubule-associated protein MAST/Orbit as an example for the usefulness of the technique.

No MeSH data available.


Related in: MedlinePlus