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An inducible system for expression and validation of the specificity of short hairpin RNA in mammalian cells.

Ma HT, On KF, Tsang YH, Poon RY - Nucleic Acids Res. (2007)

Bottom Line: Conversely, the rescue protein can be activated after the endogenous protein is completely repressed.This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth.This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.

ABSTRACT
RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.

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Manifestation of knockdown phenotypes after the removal of the rescue cyclin A or MAD2. (A) Cyclin A is silenced by shRNA in the whole cell population. Stable cyclin A/shRNA-expressing cells (clone 3) were either mock-treated or treated with doxycycline for 48 h. The cells were then fixed, stained with a monoclonal antibody against cyclin A, and processed for flow cytometry analysis. The parental HtTA1 cells were also analyzed as a control. (B) Cyclin A is repressed by shRNA throughout the cell cycle. Stable cyclin A/shRNA-expressing cells were either mock-treated or exposed to doxycycline for 48 h. The cells were then fixed, stained with a monoclonal antibody against cyclin A and propidium iodide, and subjected to bivariate flow cytometry analysis. The parental HtTA1 cells were also analyzed as a control. The positions of the 2N and 4N DNA contents are indicated. (C) Knockdown of cyclin A delays the cell cycle at G2/M. Stable cyclin A/shRNA-expressing cells were either mock-treated or treated with doxycycline for 48 h. The cells were then fixed, stained with propidium iodide, and processed for flow cytometry analysis. The parental HtTA1 cells were also analyzed as a control. The positions of the 2N and 4N DNA contents are indicated. (D) Knockdown of MAD2 abolishes the spindle-assembly checkpoint. Cells were transfected with MAD2/shRNA in pKAR1. Plasmids expressing GFP-histone H2B constitutionally were cotransfected to allow the identification of transfected cells. The cells treated with nocodazole, and were either mock-treated or treated with doxycycline for 44 h. The cells were then fixed, stained with propidium iodide, and processed for flow cytometry analysis of the GFP-positive cells. The positions of the 2N, 4N and 8N DNA contents are indicated.
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Figure 6: Manifestation of knockdown phenotypes after the removal of the rescue cyclin A or MAD2. (A) Cyclin A is silenced by shRNA in the whole cell population. Stable cyclin A/shRNA-expressing cells (clone 3) were either mock-treated or treated with doxycycline for 48 h. The cells were then fixed, stained with a monoclonal antibody against cyclin A, and processed for flow cytometry analysis. The parental HtTA1 cells were also analyzed as a control. (B) Cyclin A is repressed by shRNA throughout the cell cycle. Stable cyclin A/shRNA-expressing cells were either mock-treated or exposed to doxycycline for 48 h. The cells were then fixed, stained with a monoclonal antibody against cyclin A and propidium iodide, and subjected to bivariate flow cytometry analysis. The parental HtTA1 cells were also analyzed as a control. The positions of the 2N and 4N DNA contents are indicated. (C) Knockdown of cyclin A delays the cell cycle at G2/M. Stable cyclin A/shRNA-expressing cells were either mock-treated or treated with doxycycline for 48 h. The cells were then fixed, stained with propidium iodide, and processed for flow cytometry analysis. The parental HtTA1 cells were also analyzed as a control. The positions of the 2N and 4N DNA contents are indicated. (D) Knockdown of MAD2 abolishes the spindle-assembly checkpoint. Cells were transfected with MAD2/shRNA in pKAR1. Plasmids expressing GFP-histone H2B constitutionally were cotransfected to allow the identification of transfected cells. The cells treated with nocodazole, and were either mock-treated or treated with doxycycline for 44 h. The cells were then fixed, stained with propidium iodide, and processed for flow cytometry analysis of the GFP-positive cells. The positions of the 2N, 4N and 8N DNA contents are indicated.

Mentions: Although the total cyclin A was reduced to a very low level when both the endogenous and the rescue cyclin A were repressed, it is conceivable that a minor portion of cells still expressed high levels of cyclin A. To examine this possibility, the abundance of cyclin A in individual cells was determined with flow cytometry. Several lines of evidence indicate that cyclin A is actively degraded during mitosis and G1 phase (24). In agreement with this, two populations of cells with different cyclin A levels were detected with flow cytometry (Figure 6A). Doxycycline reduced the expression of cyclin A in the entire population, suggesting that cyclin A was not only eliminated in selected cells. After staining with propidium iodide, bivariate analysis further indicated that cyclin A was reduced in different phases of the cell cycle (Figure 6B).Figure 6.


An inducible system for expression and validation of the specificity of short hairpin RNA in mammalian cells.

Ma HT, On KF, Tsang YH, Poon RY - Nucleic Acids Res. (2007)

Manifestation of knockdown phenotypes after the removal of the rescue cyclin A or MAD2. (A) Cyclin A is silenced by shRNA in the whole cell population. Stable cyclin A/shRNA-expressing cells (clone 3) were either mock-treated or treated with doxycycline for 48 h. The cells were then fixed, stained with a monoclonal antibody against cyclin A, and processed for flow cytometry analysis. The parental HtTA1 cells were also analyzed as a control. (B) Cyclin A is repressed by shRNA throughout the cell cycle. Stable cyclin A/shRNA-expressing cells were either mock-treated or exposed to doxycycline for 48 h. The cells were then fixed, stained with a monoclonal antibody against cyclin A and propidium iodide, and subjected to bivariate flow cytometry analysis. The parental HtTA1 cells were also analyzed as a control. The positions of the 2N and 4N DNA contents are indicated. (C) Knockdown of cyclin A delays the cell cycle at G2/M. Stable cyclin A/shRNA-expressing cells were either mock-treated or treated with doxycycline for 48 h. The cells were then fixed, stained with propidium iodide, and processed for flow cytometry analysis. The parental HtTA1 cells were also analyzed as a control. The positions of the 2N and 4N DNA contents are indicated. (D) Knockdown of MAD2 abolishes the spindle-assembly checkpoint. Cells were transfected with MAD2/shRNA in pKAR1. Plasmids expressing GFP-histone H2B constitutionally were cotransfected to allow the identification of transfected cells. The cells treated with nocodazole, and were either mock-treated or treated with doxycycline for 44 h. The cells were then fixed, stained with propidium iodide, and processed for flow cytometry analysis of the GFP-positive cells. The positions of the 2N, 4N and 8N DNA contents are indicated.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Manifestation of knockdown phenotypes after the removal of the rescue cyclin A or MAD2. (A) Cyclin A is silenced by shRNA in the whole cell population. Stable cyclin A/shRNA-expressing cells (clone 3) were either mock-treated or treated with doxycycline for 48 h. The cells were then fixed, stained with a monoclonal antibody against cyclin A, and processed for flow cytometry analysis. The parental HtTA1 cells were also analyzed as a control. (B) Cyclin A is repressed by shRNA throughout the cell cycle. Stable cyclin A/shRNA-expressing cells were either mock-treated or exposed to doxycycline for 48 h. The cells were then fixed, stained with a monoclonal antibody against cyclin A and propidium iodide, and subjected to bivariate flow cytometry analysis. The parental HtTA1 cells were also analyzed as a control. The positions of the 2N and 4N DNA contents are indicated. (C) Knockdown of cyclin A delays the cell cycle at G2/M. Stable cyclin A/shRNA-expressing cells were either mock-treated or treated with doxycycline for 48 h. The cells were then fixed, stained with propidium iodide, and processed for flow cytometry analysis. The parental HtTA1 cells were also analyzed as a control. The positions of the 2N and 4N DNA contents are indicated. (D) Knockdown of MAD2 abolishes the spindle-assembly checkpoint. Cells were transfected with MAD2/shRNA in pKAR1. Plasmids expressing GFP-histone H2B constitutionally were cotransfected to allow the identification of transfected cells. The cells treated with nocodazole, and were either mock-treated or treated with doxycycline for 44 h. The cells were then fixed, stained with propidium iodide, and processed for flow cytometry analysis of the GFP-positive cells. The positions of the 2N, 4N and 8N DNA contents are indicated.
Mentions: Although the total cyclin A was reduced to a very low level when both the endogenous and the rescue cyclin A were repressed, it is conceivable that a minor portion of cells still expressed high levels of cyclin A. To examine this possibility, the abundance of cyclin A in individual cells was determined with flow cytometry. Several lines of evidence indicate that cyclin A is actively degraded during mitosis and G1 phase (24). In agreement with this, two populations of cells with different cyclin A levels were detected with flow cytometry (Figure 6A). Doxycycline reduced the expression of cyclin A in the entire population, suggesting that cyclin A was not only eliminated in selected cells. After staining with propidium iodide, bivariate analysis further indicated that cyclin A was reduced in different phases of the cell cycle (Figure 6B).Figure 6.

Bottom Line: Conversely, the rescue protein can be activated after the endogenous protein is completely repressed.This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth.This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.

ABSTRACT
RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.

Show MeSH