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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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Regulation of cyclin A expression in stable cyclin A/shRNA-expressing cells. (A) Dose-dependent repression of cyclin A by doxycycline in stable cyclin A/shRNA-expressing cells. A cyclin A/shRNA-stable cell line (clone 11) was treated with different doses of doxycycline (from lanes 2–11: 1.3, 2.6, 3.3, 4.4, 6.6, 13.3, 40, 400 and 2000 ng/ml) for 48 h. Cell-free extracts were prepared and the expression of cyclin A was detected by immunoblotting. Extracts from control cells were loaded in lane 1 and CDC2 analysis was included to assess protein loading and transfer. (B) Time-dependent repression of cyclin A by doxycycline in stable cyclin A/shRNA-expressing cells. A cyclin A/shRNA-stable cell line (clone 3) was exposed to 2 µg/ml of doxycycline and harvested at the indicated time points. Cell-free extracts were prepared and were subjected to immunoblotting for cyclin A and cyclin B1. Extracts from control cells were loaded in lane 1 and uniform loading of lysates was confirmed by immunoblotting for CDC2.
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Figure 5: Regulation of cyclin A expression in stable cyclin A/shRNA-expressing cells. (A) Dose-dependent repression of cyclin A by doxycycline in stable cyclin A/shRNA-expressing cells. A cyclin A/shRNA-stable cell line (clone 11) was treated with different doses of doxycycline (from lanes 2–11: 1.3, 2.6, 3.3, 4.4, 6.6, 13.3, 40, 400 and 2000 ng/ml) for 48 h. Cell-free extracts were prepared and the expression of cyclin A was detected by immunoblotting. Extracts from control cells were loaded in lane 1 and CDC2 analysis was included to assess protein loading and transfer. (B) Time-dependent repression of cyclin A by doxycycline in stable cyclin A/shRNA-expressing cells. A cyclin A/shRNA-stable cell line (clone 3) was exposed to 2 µg/ml of doxycycline and harvested at the indicated time points. Cell-free extracts were prepared and were subjected to immunoblotting for cyclin A and cyclin B1. Extracts from control cells were loaded in lane 1 and uniform loading of lysates was confirmed by immunoblotting for CDC2.

Mentions: To determine if fine adjustment of cyclin A expression can be achieved in the stable cell lines, cells were treated with different concentrations of doxycycline before harvested. Figure 5A shows that a range of cyclin A expression, from an undetectable level to a highly overexpressed level, was obtained by varying the doxycycline concentration. The expression of FLAG-cyclin A could be turned off relatively rapidly (Figure 5B). Our conclusion is that while the method is rather robust, the precise dose and time of doxycycline adopted for fine adjustment of the rescue protein will have to be determined empirically (which depends on the half-life of the protein and its levels relative to the endogenous protein).Figure 5.


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)

Regulation of cyclin A expression in stable cyclin A/shRNA-expressing cells. (A) Dose-dependent repression of cyclin A by doxycycline in stable cyclin A/shRNA-expressing cells. A cyclin A/shRNA-stable cell line (clone 11) was treated with different doses of doxycycline (from lanes 2–11: 1.3, 2.6, 3.3, 4.4, 6.6, 13.3, 40, 400 and 2000 ng/ml) for 48 h. Cell-free extracts were prepared and the expression of cyclin A was detected by immunoblotting. Extracts from control cells were loaded in lane 1 and CDC2 analysis was included to assess protein loading and transfer. (B) Time-dependent repression of cyclin A by doxycycline in stable cyclin A/shRNA-expressing cells. A cyclin A/shRNA-stable cell line (clone 3) was exposed to 2 µg/ml of doxycycline and harvested at the indicated time points. Cell-free extracts were prepared and were subjected to immunoblotting for cyclin A and cyclin B1. Extracts from control cells were loaded in lane 1 and uniform loading of lysates was confirmed by immunoblotting for CDC2.
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Related In: Results  -  Collection

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Figure 5: Regulation of cyclin A expression in stable cyclin A/shRNA-expressing cells. (A) Dose-dependent repression of cyclin A by doxycycline in stable cyclin A/shRNA-expressing cells. A cyclin A/shRNA-stable cell line (clone 11) was treated with different doses of doxycycline (from lanes 2–11: 1.3, 2.6, 3.3, 4.4, 6.6, 13.3, 40, 400 and 2000 ng/ml) for 48 h. Cell-free extracts were prepared and the expression of cyclin A was detected by immunoblotting. Extracts from control cells were loaded in lane 1 and CDC2 analysis was included to assess protein loading and transfer. (B) Time-dependent repression of cyclin A by doxycycline in stable cyclin A/shRNA-expressing cells. A cyclin A/shRNA-stable cell line (clone 3) was exposed to 2 µg/ml of doxycycline and harvested at the indicated time points. Cell-free extracts were prepared and were subjected to immunoblotting for cyclin A and cyclin B1. Extracts from control cells were loaded in lane 1 and uniform loading of lysates was confirmed by immunoblotting for CDC2.
Mentions: To determine if fine adjustment of cyclin A expression can be achieved in the stable cell lines, cells were treated with different concentrations of doxycycline before harvested. Figure 5A shows that a range of cyclin A expression, from an undetectable level to a highly overexpressed level, was obtained by varying the doxycycline concentration. The expression of FLAG-cyclin A could be turned off relatively rapidly (Figure 5B). Our conclusion is that while the method is rather robust, the precise dose and time of doxycycline adopted for fine adjustment of the rescue protein will have to be determined empirically (which depends on the half-life of the protein and its levels relative to the endogenous protein).Figure 5.

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